Enzymatic production of hexoses

ABSTRACT

Disclosed herein are methods of producing hexoses from saccharides by enzymatic processes. The methods utilize fructose 6-phosphate and at least one enzymatic step to convert it to a hexose.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 16/493,519, filed Sep. 12, 2019; which is a national phase of PCTInternational Application No. PCT/US2018/022185, filed on Mar. 13, 2018;which claims priority to U.S. Application No. 62/470,605, filed on Mar.13, 2017, U.S. Application No. 62/470,620, filed on Mar. 13, 2017, U.S.Application No. 62/482,148, filed on Apr. 5, 2017, and U.S. ApplicationNo. 62/480,798, filed on Apr. 3, 2017, which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The Sequence Listing submitted herewith as an ASCII text file(2022-05-26_Divisional_Sequence_Listing.txt, created on Apr. 27, 2022,50533 bytes) via EFS-Web is hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to preparation of hexose monosaccharides. Morespecifically, the invention relates to methods of preparing a D-hexose(or hexose) from saccharides (e.g., polysaccharides, oligosaccharides,disaccharides, sucrose, D-glucose, and D-fructose) including a step inwhich fructose 6-phosphate is converted to the hexose by one or moreenzymatic steps.

BACKGROUND

Hexoses are monosaccharides with six carbon atoms. Hexoses can beclassified by functional group, with aldohexoses having an aldehyde atposition 1, and ketohexoses having that ketone at position 2.Aldohexoses (or aldoses) include allose, altrose, glucose, gulose,galactose, idose, talose, and mannose. Ketohexoses (or ketoses) includepsicose (allulose), fructose, tagatose, and sorbose. Various aspects ofthese aldohexoses and ketohexoses are mentioned in the followingparagraphs.

For example, D-allose (allose hereafter) is a low-calorie, naturalsweetener that has {tilde over ( )}80% the sweetness of sucrose and isdescribed as a noncaloric sweetening and bulking agent. It is anaturally occurring monosaccharide hexose that is present in only smallamounts in specific shrubs and algae. Allose boasts several potentialmedical and agriculture benefits including cryoprotective,anti-oxidative, anti-hypertensive, immunosuppressive, anti-inflammatory,anti-tumor, and anti-cancer activities. It also has similarfunctionality in foods and beverages to sucrose. As such, allose clearlyhas a variety of applications in the food and beverage industries.However, due to allose's high selling prices, its use as a sweetener hasbeen limited.

Currently allose is produced predominantly through the enzymaticisomerization of D-psicose (WO 2014069537). Overall, the method suffersbecause of higher feedstock cost, the costly separation of allose fromD-psicose, and relatively low product yields ({tilde over ( )}23%).

Altrose is another unnatural aldohexose and C-3 epimer of mannose.D-Altrose ((2S,3R,4R,5R)-2,3,4,5,6-Pentahydroxyhexanal) can be used as asubstrate to identify, differentiate and characterize aldose isomerasessuch as L-fucose isomerase from Caldicellulosiruptor saccharolydcus andd-Arabinose isomerase (d-AI) from Bacillus pallidus (B. pallidus) andKlebsiella pneumoniae. Recently, sugar chains such as oligosaccharidesand polysaccharides, which perform functions useful as a physiologicallyactive substance, have attracted attention in the field of finechemicals such as medicines and agricultural chemicals. Presently, theobjects of researches on the sugar chain are restricted to thoseconsisting of monosaccharides present in nature in large amounts andreadily available to researchers, such as D-glucose, D-mannose andD-galactose. However, it is expected that various monosaccharides otherthan those present in nature will be required in the future in researchon the synthesis of sugar chains performing more useful functions. Underthe circumstances, it is highly significant and necessary to develop amethod which permits preparing D-altrose, which is a rare sugardifficult to obtain, in high yield while diminishing the number oftreating steps. U.S. Pat. No. 5,410,038.

D-Gulose is useful, for example, as an excipient, a chelating agent, apharmaceutical intermediate, a cleaning agent for glass and metals, afood additive, and as an additive for detergents. U.S. Pat. No.5,215,591.

D-galactose (galactose hereafter) is a natural sweetener that has {tildeover ( )}33% the sweetness of sucrose and is described as a nutritivesweetener. It is a naturally occurring monosaccharide hexose that ispresent in dairy products, legumes, grains, nuts, tubers and vegetables.Galactose is used by the baking industry to limit tartness and acidityin foods. Also, it is used as an energy source to increase endurance inthe exercise supplement industry. In the pharmaceutical industry it isan intermediate for several medicines and is also used as a cellmetabolism modulator in the optimization of protein therapeuticsbioproduction. Additionally, galactose has been shown to be effective asa control agent against plant disease caused by certain plant pathogens,such as those affecting cucumber, carrot, potato and tomato plants. Dueto dietary concerns (e.g. veganism) and health concerns (e.g. BSEdisease) non-animal sources of galactose are of interest to industry. Assuch, galactose clearly has a variety of applications in the food,beverage, exercise, agriculture, and pharmaceutical industries. However,due to galactose's high selling prices, its use has been limited.

Galactose is produced predominantly through the hydrolysis of lactose(WO 2005039299A3). This method is less desirable due to a more costlyfeed stock and the expensive separation of glucose from galactose.Alternatively, galactose can be produced via the hydrolysis ofplant-based biomass (WO 2005001145A1). This method suffers from thecostly separation of galactose from the multiple other sugars releasedduring biomass hydrolysis (e.g. xylose, arabinose, mannose, glucose, andrhamnose) and low yields ({tilde over ( )}4.6% of the dry mass of commonbiomass sources is galactose).

Idose is not found in nature, but its uronic acid, iduronic acid, isimportant. It is a component of dermatan sulfate and heparan sulfate,which are glycosaminoglycans.

(https://en.wikipedia.org/wiki/Idose-accessed 3/7/18).

Talose is an unnatural aldohexose that is soluble in water and slightlysoluble in methanol. It is a C-2 epimer of galactose and C-4 epimer ofmannose. Talose can be used as a substrate to identify, differentiate,and characterize ribose-5-phosphate isomerase(s) of Clostridia.

D-mannose (mannose hereafter) is a mildly sweet, naturally-occurringmonosaccharide that is found in many fruits, vegetables, plantmaterials, and even the human body. Mannose boasts multiple healthbenefits and pharmaceutical applications. For example, mannose can beused to treat carbohydrate-deficient glycoprotein syndrome type 1b and,more commonly, urinary tract infections. Furthermore, mannose is averified prebiotic, does not raise blood glucose levels, and showsanti-inflammatory properties. Additionally, it has been shown to enhancecarcass yields in pigs and is a widely used auxiliary moisturizing agentfor skin-care products. As such, mannose has a variety of applicationsin the pharmaceutical, cosmetic, beverage, food product, dairy,confectionery, and livestock industries. However, due to mannose's highselling prices, its use in everyday products has been limited.

Mannose is primarily produced through extraction from plants. Commonmethods include acid hydrolysis, thermal hydrolysis, enzymatichydrolysis, microbial fermentation hydrolysis, and mixtures thereof.Less common methods include chemical and biological transformations.Overall, these methods are problematice due to harsh conditions, highcapital expenditures, higher feedstock cost, costly separation ofmannose from isomerization reactions, and relatively low product yields(15-35%).

D-allulose (also known as D-psicose) (psicose hereafter) is alow-calorie, natural sweetener that has 70% the sweetness of sucrose,but only 10% of the calories. It is a naturally occurring monosaccharidehexose that is present in only small amounts in wheat and other plants.Psicose was approved as a food additive by the Food and DrugAdministration (FDA) in 2012, which designated it as generallyrecognized as safe (GRAS). However, due to psicose's high sellingprices, its use as a sweetener has been limited. Psicose boasts a myriadof health benefits: it is low-calorie (10% of sucrose); it has a verylow glycemic index of 1; it is fully absorbed in the small intestine butnot metabolized and instead secreted in urine and feces; it helpsregulate blood sugar by inhibiting alpha-amylase, sucrase and maltase;and it has similar functionality in foods and beverages as sucrose. Assuch, psicose clearly has a variety of applications in the food andbeverage industries.

Currently psicose is produced predominantly through the enzymaticisomerization of fructose (WO 2014049373). Overall, the method exhibitshigher feedstock cost, the costly separation of psicose from fructose,and relatively low product yields.

Fructose is a simple ketonic monosaccharide found in many plants, whereit is often bonded to glucose to form the disaccharide, sucrose.Commercially, fructose is derived from sugar cane, sugar beets, andmaize. The primary reason that fructose is used commercially in foodsand beverages, besides its low cost, is its high relative sweetness. Itis the sweetest of all naturally occurring carbohydrates. Fructose isalso found in the manufactured sweetener, high-fructose corn syrup(HFCS), which is produced by treating corn syrup with enzymes,converting glucose into fructose.

(https://en.wikipedia.org/wiki/Fructose#Physical_and_functional_properties—accessed3/7/18).

D-tagatose (tagatose hereafter) is a low-calorie, natural sweetener thathas 92% the sweetness of sucrose, but only 38% of the calories. It is anaturally occurring monosaccharide hexose that is present in only smallamounts in fruits, cacao, and dairy products. Tagatose was approved as afood additive by the Food and Drug Administration (FDA) in 2003, whichdesignated it as generally recognized as safe (GRAS). However, due totagatose's high selling prices, its use as a sweetener has been limited.Tagatose boasts a myriad of health benefits: it is non-cariogenic; it islow-calorie; it has a very low glycemic index of 3; it attenuates theglycemic index of glucose by 20%; it can lower average blood glucoselevels; it helps prevent cardiovascular disease, strokes, and othervascular diseases by raising high-density lipoprotein (HDL) cholesterol;and it is a verified prebiotic and antioxidant. Lu et al., Tagatose, aNew Antidiabetic and Obesity Control Drug, Diabetes Obes. Metab. 10(2):109-34 (2008). As such, tagatose clearly has a variety of applicationsin the pharmaceutical, biotechnological, academic, food, beverage,dietary supplement, and grocer industries.

Tagatose is produced predominantly through the hydrolysis of lactose bylactase or acid hydrolysis to form D-glucose and D-galactose (WO2011150556, CN 103025894, U.S. Pat. Nos. 5,002,612, 6,057,135, and8,802,843). The D-galactose is then isomerized to D-tagatose eitherchemically by calcium hydroxide under alkaline conditions orenzymatically by L-arabinose isomerase under pH neutral conditions. Thefinal product is isolated by a combination of filtration and ionexchange chromatography. This process is performed in several tanks orbioreactors. Overall, the method is disadvantageous because of thecostly separation of other sugars (e.g., D-glucose, D-galactose, andunhydrolyzed lactose) and low product yields. Several methods viamicrobial cell fermentation are being developed, but none have beenproven to be a practical alternative due to their dependence on costlyfeedstock (e.g., galactitol and D-psicose), low product yields, andcostly separation.

Sorbose ((3R,4S,5R)-1,3,4,5,6-pentahydroxyhexan-2-one) is a ketohexosethat has a sweetness equivalent to sucrose (table sugar), and it is aplant metabolite that has been found to naturally occur in grapes insmall quantities. D-sorbose has been determined to be effective as acontrol agent of plant diseases caused by: Pseudomonas syringae pv.lachrymans and Ralstonia solanacearum. United States Patent ApplicationPublication No. 2016/0037768.

There is a need to develop cost-effective synthetic pathways forhigh-yield production of the hexoses such as the aldohexoses andaldoketoses discussed above where at least one step of the processesinvolves an energetically favorable chemical reaction. Furthermore,there is a need for production processes where the process steps can beconducted in one tank or bioreactor and/or where costly separation stepsare avoided or eliminated. There is also a need for processes of hexoseproduction that can be conducted at a relatively low concentration ofphosphate, where phosphate can be recycled, and/or the process does notrequire using adenosine triphosphate (ATP) as an added source ofphosphate. There is also a need for hexose production pathways that donot require the use of the costly nicotinamide adenosine dinucleotide(NAD(P)(H)) coenzyme in any of the reaction steps.

SUMMARY OF THE INVENTION

The inventions described herein generally relate to processes forpreparing hexoses from saccharides by enzymatic conversion. Theinventions also relate to hexoses prepared by any of the processesdescribed herein.

More specifically, the invention relates to processes for preparing ahexose, selected from allose, mannose, galactose, fructose, altrose,talose, sorbose, gulose and idose, from a saccharide, the processcomprising: converting fructose 6-phosphate (F6P) to the hexosecatalyzed by one or more enzymes selected from an isomerase, anepimerase, and a hexose-specific phosphatase and mixtures thereof.

A process of the invention for the production of allose comprisesconverting the F6P to psicose 6-phosphate (P6P) catalyzed by psicose6-phosphate 3-epimerase (P6PE); converting the P6P to allose 6-phosphate(A6P) catalyzed by allose 6-phosphate isomerase (A6PI); and convertingthe A6P to allose catalyzed by allose 6-phosphate phosphatase (A6PP).

A process of the invention for the production of mannose comprisesconverting the F6P to mannose 6-phosphate (M6P) catalyzed by mannose6-phosphate isomerase (M6PI) or phosphoglucose/phosphomannose isomerase(PGPMI); and converting the M6P to mannose catalyzed by mannose6-phosphate phosphatase (M6PP).

A process of the invention for the production of galactose comprisesconverting the F6P to tagaose 6-phosphate (T6P) catalyzed by fructose6-phosphate 4-epimerase (F6PE); converting the T6P to galactose6-phosphate (Gal6P) catalyzed by galactose 6-phosphate isomerase(Gal6PI); and converting the Gal6P to galactose catalyzed by galactose6-phosphate phosphatase (Gal6PP).

A process of the invention for the production of fructose comprisesconverting the F6P to fructose catalyzed by fructose 6-phosphatephosphatase (F6PP).

A process of the invention for the production of altrose comprisesconverting the F6P to converting the F6P to P6P catalyzed by P6PE;converting the P6P to altrose 6-phosphate (Alt6P) catalyzed by altrose6-phosphate isomerase (Alt6PI); and converting the Alt6P produced toaltrose catalyzed by altrose 6-phosphate phosphatase (Alt6PP).

A process of the invention for the production of talose comprisesconverting the F6P to T6P catalyzed by F6PE; converting the T6P totalose 6-phosphate (Tal6P) catalyzed by talose 6-phosphate isomerase(Tal6PI); and converting the Tal6P to talose catalyzed by talose6-phosphate phosphatase (Tal6PP).

A process of the invention for the production of sorbose comprisesconverting the F6P to T6P catalyzed by F6PE; converting the T6P tosorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase(S6PE); and converting the S6P to sorbose catalyzed by sorbose6-phosphate phosphatase (S6PP).

A process of the invention for the production of gulose comprisesconverting the F6P to T6P catalyzed by F6PE; converting the S6P togulose 6-phosphate (Gul6P) catalyzed by gulose 6-phosphate isomerase(Gul6PI); and converting the Gul6P to gulose catalyzed by gulose6-phosphate phosphatase (Gul6PP).

A process of the invention for the production of gulose comprisesconverting the F6P to T6P catalyzed by F6PE; converting the T6P tosorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase(S6PE); converting the S6P to idose 6-phosphate (16P) catalyzed by idose6-phosphate isomerase (16PI); and converting the 16P to idose catalyzedby idose 6-phosphate phosphatase (16PP).

The processes of hexose production according to the invention caninvolve a step of converting glucose 6-phosphate (G6P) to the F6P,wherein the step is catalyzed by phosphoglucose isomerase (PGI). Theprocesses can also comprise the step of converting glucose 1-phosphate(G1P) to the G6P, wherein the step is catalyzed by phosphoglucomutase(PGM). Additionally, the processes according to the invention mayfurther comprise the step of converting a saccharide to the G1P, wherethe step is catalyzed by at least one enzyme, and the saccharide isselected from the group consisting of a starch or derivative thereof,cellulose or a derivative thereof, and sucrose.

The enzyme or enzymes used in the step of converting a saccharide to theG1P in the processes according to the invention can be alpha-glucanphosphorylase (αGP), maltose phosphorylase, sucrose phosphorylase,cellodextrin phosphorylase, cellobiose phosphorylase, and/or cellulosephosphorylase, and mixtures thereof. When the saccharide is starch or astarch derivative, the derivative may be selected from the groupconsisting of amylose, amylopectin, soluble starch, amylodextrin,maltodextrin, maltose, and glucose, and mixtures thereof.

Some processes according to the invention, may further comprise the stepof converting starch to a starch derivative, where the starch derivativeis prepared by enzymatic hydrolysis of starch or by acid hydrolysis ofstarch. Also, 4-glucan transferase (4GT) can be added to the processes.4GT can be used to increase hexose yields by recycling the degradationproducts glucose, maltose, and maltotriose into longermaltooligosaccharides; which can be phosphorolytically cleaved by αGP toyield G1P.

Where the processes use a starch derivative, the starch derivative canbe prepared by enzymatic hydrolysis of starch catalyzed by isoamylase,pullulanase, alpha-amylase, or their combination.

The process according to the inventions can also comprise the step ofconverting fructose to the F6P, wherein the step is catalyzed by atleast one enzyme and, optionally, the step of converting sucrose to thefructose, wherein the step is catalyzed by at least one enzyme.

Furthermore, the processes of producing a hexose according to theinventions can comprise the step of converting glucose to the G6P, wherethe step is catalyzed by at least one enzyme, and, optionally, the stepof converting sucrose to the glucose that is catalyzed by at least oneenzyme.

The steps in each of the processes of hexose synthesis according to theinvention can be conducted at a temperature ranging from about 40° C. toabout 90° C. and at a pH ranging from about 5.0 to about 8.0. They maybe conducted for about 8 hours to about 48 hours.

The steps of the processes according to the inventions can be conductedin a single bioreactor. The steps can also be conducted in a pluralityof bioreactors arranged in series.

The enzymatic process steps of the inventions may be conducted ATP-freeand/or NAD(P)(H)-free. The steps can be carried out at a phosphateconcentration ranging from about 0.1 mM to about 150 mM. The phosphateused in the phosphorylation and dephosphorylation steps of the processesaccording to the inventions can be recycled. At least one step of theprocesses may involve an energetically favorable chemical reaction.

The invention also relates to allose, mannose, galactose, fructose,altrose, talose, sorbose, gulose and idose produced by these processes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to allose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; P6PE, psicose 6-phosphate 3-epimerase; A6P1,allose 6-phosphate isomerase; A6PP, allose 6-phosphate phosphatase.

FIG. 2 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to mannose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; PGPMI, bifunctional phosphoglucose/phosphomannoseisomerase; M6P1, mannose 6-phosphate isomerase; M6PP, mannose6-phosphate phosphatase.

FIG. 3 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to galactose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; F6PE, fructose 6-phosphate isomerase; Gal6PI,galactose 6-phosphate isomerase; Gal6PP, galactose 6-phosphatephosphatase.

FIG. 4 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to fructose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; F6PP, fructose 6-phosphate phosphatase.

FIG. 5 is a schematic diagram showing an enzymatic pathway convertingsucrose to fructose. The following abbreviations are used: SP, sucrosephosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase;F6PP, fructose 6-phosphate phosphatase.

FIG. 6 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to altrose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; P6PE, psicose 6-phosphate epimerase; Alt6PI,altrose 6-phosphate isomerase; Alt6PP, altrose 6-phosphate phosphatase.

FIG. 7 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to talose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; Tal6PI,talose 6-phosphate isomerase; Tal6PP, talose 6-phosphate phosphatase.

FIG. 8 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to sorbose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; S6PE,sorbose 6-phosphate epimerase; S6PP, sorbose 6-phosphate phosphatase.

FIG. 9 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to gulose. The following abbreviationsare used: IA, isoamylase; PA, pullulanase; αGP, alpha-glucanphosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; S6PE,sorbose 6-phosphate epimerase; Gul6PI, gulose 6-phosphate isomerase;Gul6PP, gulose 6-phosphate phosphatase.

FIG. 10 is a schematic diagram showing an enzymatic pathway convertingstarch or its derived products to idose. The following abbreviations areused: IA, isoamylase; PA, pullulanase; αGP, alpha-glucan phosphorylaseor starch phosphorylase; MP, maltose phosphorylase; PGM,phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; S6PE,sorbose 6-phosphate epimerase; 16P1, idose 6-phosphate isomerase; 16PP,idose 6-phosphate phosphatase.

FIG. 11 shows the Reaction Gibbs Energy between intermediates based onformation Gibbs energy for the conversion of glucose 1-phosphate toanother hexose.

DESCRIPTION OF THE INVENTION

The inventions described herein provide enzymatic pathways, orprocesses, for synthesizing hexoses with a high product yield, whilegreatly decreasing the product separation costs and hexose productioncosts. Also described herein are hexoses produced by these process.

Processes according to the invention for preparing a hexose from asaccharide, comprise: converting fructose 6-phosphate (F6P) to thehexose, catalyzed by one or more enzymes, wherein the hexose is selectedfrom the group consisting of allose, mannose, galactose, fructose,altrose, talose, sorbose, gulose and idose; and wherein the enzymes areselected from the group consisting of an isomerase, an epimerase, and ahexose-specific phosphatase, and mixtures thereof.

One of the important advantages of the processes of the invention isthat the process steps can be conducted in a single bioreactor orreaction vessel. Alternatively, the steps can also be conducted in aplurality of bioreactors, or reaction vessels, that are arranged inseries.

Phosphate ions produced during the dephosphorylation step can then berecycled in the process step of converting a saccharide to G1P,particularly when all process steps are conducted in a single bioreactoror reaction vessel. The ability to recycle phosphate in the disclosedprocesses allows for non-stoichiometric amounts of phosphate to be used,which keeps reaction phosphate concentrations low. This affects theoverall pathway and the overall rate of the processes, but does notlimit the activity of the individual enzymes and allows for overallefficiency of the hexose making processes.

For example, reaction phosphate concentrations in each of the processescan range from about 0.1 mM to about 300 mM, from about 0 mM to about150 mM, from about 1 mM to about 50 mM, preferably from about 5 mM toabout 50 mM, or more preferably from about 10 mM to about 50 mM. Forinstance, the reaction phosphate concentration in each of the processescan be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM,about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM,about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

Low phosphate concentration results in decreased production costs due tolow total phosphate and thus lowered cost of phosphate removal. It alsoprevents inhibition of phosphatases by high concentrations of freephosphate and decreases the potential for phosphate pollution.

Furthermore, each of the processes disclosed herein can be conductedwithout added ATP as a source of phosphate, i.e., ATP-free. Each of theprocesses can also be conducted without having to add NAD(P)(H), i.e.,NAD(P)(H)-free. Other advantages also include the fact that at least onestep of the disclosed processes for making a hexose involves anenergetically favorable chemical reaction.

Examples of the enzymes used to convert a saccharide to G1P includealpha-glucan phosphorylase (αGP, EC 2.4.1.1), maltose phosphorylase (MP,EC 2.4.1.8), cellodextrin phosphorylase (CDP, EC 2.4.1.49), cellobiosephosphorylase (CBP, EC 2.4.1.20), cellulose phosphorylase, sucrosephosphorylase (SP, EC 2.4.1.7), and a combination thereof. The choice ofthe enzyme or enzyme combination depends on the saccharide used in theprocess.

The saccharides used for generating G1P can be polysaccharides,oligosaccharides, and/or disaccharides. For example, the saccharide canbe starch, one or more derivatives of starch, cellulose, one or morederivatives of cellulose, sucrose, one or more derivatives of sucrose,or a combination thereof.

Starch is the most widely used energy storage compound in nature and ismostly stored in plant seeds. Natural starch contains linear amylose andbranched amylopectin. Examples of starch derivatives include amylose,amylopectin, soluble starch, amylodextrin, maltodextrin, maltose,fructose, and glucose. Examples of cellulose derivatives includepretreated biomass, regenerated amorphous cellulose, cellodextrin,cellobiose, fructose, and glucose. Sucrose derivatives include fructoseand glucose.

Methods of preparing F6P from starch and its derivatives, cellulose andits derivatives, and sucrose and its derivatives can be found, forexample in International Patent Application Publication No. WO2017/059278.

The derivatives of starch can be prepared by enzymatic hydrolysis ofstarch or by acid hydrolysis of starch. Specifically, the enzymatichydrolysis of starch can be catalyzed or enhanced by isoamylase (IA, EC.3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds; pullulanase (PA, EC.3.2.1.41), which hydrolyzes α-1,6-glucosidic bonds;4-α-glucanotransferase (4GT, EC. 2.4.1.25), which catalyzes thetransglycosylation of short maltooligosaccharides, yielding longermaltooligosaccharides; or alpha-amylase (EC 3.2.1.1), which cleavesα-1,4-glucosidic bonds.

Furthermore, derivatives of cellulose can be prepared by enzymatichydrolysis of cellulose catalyzed by cellulase mixtures, by acids, or bypretreatment of biomass.

Enzymes used to convert a saccharide to G1P may contain αGP. In thisstep, when the saccharides include starch, the G1P is generated fromstarch by αGP; when the saccharides contain soluble starch,amylodextrin, or maltodextrin, the G1P is produced from soluble starch,amylodextrin, or maltodextrin by αGP.

When the saccharides include maltose and the enzymes contain maltosephosphorylase, the G1P is generated from maltose by maltosephosphorylase. If the saccharides include sucrose, and enzymes containsucrose phosphorylase, the G1P is generated from sucrose by sucrosephosphorylase.

When the saccharides include cellobiose, and the enzymes containcellobiose phosphorylase, the G1P may be produced from cellobiose bycellobiose phosphorylase.

When the saccharides contain cellodextrins and the enzymes includecellodextrin phosphorylase, the G1P can be generated from cellodextrinsby cellodextrin phosphorylase.

In converting a saccharide to G1P, when the saccharides includecellulose, and enzymes contain cellulose phosphorylase, the G1P may begenerated from cellulose by cellulose phosphorylase.

According to the invention, a hexose can also be produced from fructose.For example, the process involves generating F6P from fructose andpolyphosphate catalyzed by polyphosphate fructokinase (PPFK); convertingF6P to T6P catalyzed by F6PE; and converting T6P to tagatose catalyzedby T6PP. The fructose can be produced, for example, by an enzymaticconversion of sucrose.

A hexose can be produced from sucrose. The process, for example,provides an in vitro synthetic pathway that includes the followingenzymatic steps: generating G1P from sucrose and free phosphatecatalyzed by sucrose phosphorylase (SP); converting G1P to G6P catalyzedby PGM; converting G6P to F6P catalyzed by PGI; converting F6P to T6Pcatalyzed by F6PE; and converting T6P to tagatose catalyzed by T6PP.

The phosphatase used in the processes of the invention is specific forthe hexose. For example, allose 6-phosphate is converted to allose byallose 6-phosphate phosphatase; mannose 6-phosphate is converted tomannose by mannose 6-phosphate phosphatase; galactose 6-phosphate isconverted to galactose by galactose 6-phosphate phosphatase; fructose6-phosphate is converted to fructose by fructose 6-phosphatephosphatase; altrose 6-phosphate is converted to altrose by altrose6-phosphate phosphatase; talose 6-phosphate is converted to talose bytalose 6-phosphate phosphatase; sorbose 6-phosphate is converted tosorbose by sorbose 6-phosphate phosphatase; gulose 6-phosphate isconverted to gulose by gulose 6-phosphate phosphatase; and idose6-phosphate is converted to idose by idose 6-phosphate phosphatase. Asused herein, specific means having a higher specfic activity for theindicated hexose over other hexoses. For instance, allose 6-phosphatephosphatase has a higher specific activity on allose 6-phosphate than,for example, sorbose 6-phosphate or talose 6-phosphate.

The phosphate ions generated during the hexose dephosphorylation stepcan then be recycled in the step of converting sucrose to G1P.Additionally, PPFK and polyphosphate can be used to increase hexoseyields by producing F6P from fructose generated by the phosphorolyticcleavage of sucrose by SP.

A process for preparing a hexose can include the following steps:generating glucose from polysaccharides and oligosaccharides byenzymatic hydrolysis or acid hydrolysis, converting glucose to G6Pcatalyzed by at least one enzyme, generating fructose frompolysaccharides and oligosaccharides by enzymatic hydrolysis or acidhydrolysis, and converting fructose to G6P catalyzed by at least oneenzyme. Examples of the polysaccharides and oligosaccharides areenumerated above. G6P may be produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The invention provides processes for converting saccharides, such aspolysaccharides and oligosaccharides in starch, cellulose, sucrose andtheir derived products, to a hexose. Artificial (non-natural) ATP-freeenzymatic pathways may be provided to convert starch, cellulose,sucrose, and their derived products to a hexose using cell-free enzymecocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to a hexose and enhancedsolubility.

Maltose phosphorylase (MP) can be used to increase hexose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease hexose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

Additionally, cellulose is the most abundant bio resource and is theprimary component of plant cell walls. Non-food lignocellulosic biomasscontains cellulose, hemicellulose, and lignin as well as other minorcomponents. Pure cellulose, including Avicel (microcrystallinecellulose), regenerated amorphous cellulose, bacterial cellulose, filterpaper, and so on, can be prepared via a series of treatments. Thepartially hydrolyzed cellulosic substrates include water-insolublecellodextrins whose degree of polymerization is more than 7,water-soluble cellodextrins with degree of polymerization of 3-6,cellobiose, glucose, and fructose.

Cellulose and its derived products can be converted to a hexose througha series of steps. The process provides an in vitro synthetic pathwaythat involves the following steps: generating G1P from cellodextrin andcellobiose and free phosphate catalyzed by cellodextrin phosphorylase(CDP) and cellobiose phosphorylase (CBP), respectively; converting G1Pto G6P catalyzed by PGM; converting G6P to F6P catalyzed by PGI. In thisprocess, the phosphate ions can be recycled by the step of convertingcellodextrin and cellobiose to G1P.

Several enzymes may be used to hydrolyze solid cellulose towater-soluble cellodextrins and cellobiose. Such enzymes includeendoglucanase and cellobiohydrolase, but not including beta-glucosidase(cellobiase).

Prior to cellulose hydrolysis and G1P generation, cellulose and biomasscan be pretreated to increase their reactivity and decrease the degreeof polymerization of cellulose chains. Cellulose and biomasspretreatment methods include dilute acid pretreatment, cellulosesolvent-based lignocellulose fractionation, ammonia fiber expansion,ammonia aqueous soaking, ionic liquid treatment, and partiallyhydrolyzed by using concentrated acids, including hydrochloric acid,sulfuric acid, phosphoric acid and their combinations.

Polyphosphate and polyphosphate glucokinase (PPGK) can be added to theprocesses according to the invention, thus increasing yields of a hexoseby phosphorylating the degradation product glucose to G6P.

A hexose can be generated from glucose. The processes for hexoseproduction may involve the steps of generating G6P from glucose andpolyphosphate catalyzed by polyphosphate glucokinase (PPGK) andconverting G6P to F6P catalyzed by PGI.

Any suitable biologically compatible buffering agent known in the artcan be used in each of the processes of the invention, such as HEPES,PBS, BIS-TRIS, MOPS, DIPSO, Trizma, etc. The reaction buffer for theprocesses according to the invention can have a pH ranging from 5.0-8.0.More preferably, the reaction buffer pH can range from about 6.0 toabout 7.3. For example, the reaction buffer pH can be 6.0, 6.2, 6.4,6.6, 6.8, 7.0, 7.2, or 7.3.

The reaction buffer can also contain metal cations. Examples of themetal ions include Mg²⁺ and Zn²⁺. As known in the art, suitable saltsmay be used to introduce the desired metal cation.

In each of the processes of the invention the reaction temperature atwhich the process steps are conducted can range from 37-95° C. Morepreferably, the steps can be conducted at a temperature ranging fromabout 40° C. to about 90° C. The temperature can be, for example, about40° C., about 45° C., about 50° C., about 55° C., about 60° C., about65° C., about 70° C., about 75° C., about 80° C., about 85° C., or about90° C. Preferably, the reaction temperature is about 50° C.

The reaction time of each of the disclosed processes can be adjusted asnecessary, and can range from about 8 hours to about 48 hours. Forexample, the reaction time can be about 16 hours, about 18 hours, about20 hours, about 22 hours, about 24 hours, about 26 hours, about 28hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours,about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46hours, or about 48 hours. More preferably, the reaction time is about 24hours.

Typically, the ratios of enzyme units used in each of the disclosedprocesses are 1:1 to 1:1:1:1:1 (depending on the number of catalyzedsteps in the process). To optimize product yields, these ratios can beadjusted in any number of combinations. For example, a ratio of3:1:1:1:1 can be used to maximize the concentration of phosphorylatedintermediates, which will result in increased activity of the downstreamreactions. Conversely, a ratio of 1:1:1:1:3 can be used to maintain arobust supply of phosphate for αGP, which will result in more efficientphosphorolytic cleavage of alpha-1,4-glycosidic bonds. A ratio ofenzymes, for example, 3:1:1:1:3 can be used to further increase thereaction rate. Therefore, the enzyme ratios, including other optionalenzymes discussed below, can be varied to increase the efficiency ofhexose production. For example, a particular enzyme may be present in anamount about 2×, 3×, 4×, 5×, etc. relative to the amount of otherenzymes.

Each of the processes according to the invention can achieve high yieldsdue to the very favorable equilibrium constant for the overall reaction.For example, FIG. 11 shows the Reaction Gibbs Energy betweenintermediates based on formation Gibbs energy for the conversion ofglucose 1-phosphate to a hexose. Reaction Gibbs Energies were generatedusing http://equilibrator.weizmann.ac.il/. Theoretically, up to 99%yields can be achieved if the starting material is completely convertedto an intermediate.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and their derivatives areless expensive feedstocks than, for example, lactose. When a hexose isproduced from lactose, glucose and other hexose(s) are separated viachromatography, which leads to higher production costs.

Also, the step of hexose dephosphorylation by a phosphatase according tothe invention is an irreversible phosphatase reaction, regardless of thefeedstock. Therefore, hexose is produced with a very high yield whileeffectively minimizing the subsequent product separation costs.

In some aspects of the invention, phosphatases to convert A6P, M6P, F6P,or Gal6P to their respective non-phosphorylated forms utilize a divalentmetal cofactor: preferably magnesium. In further aspects of theinvention the phosphatase contains but is not limited to containing aRossmanoid fold domain for catalysis; additionally but not limited tocontaining a C1 or C2 capping domain for substrate specificity;additionally but not limited to containing a D×D signature in the 1stβ-strand of the Rossmanoid fold for coordinating magnesium where thesecond Asp is a general acid/base catalyst; additionally but not limitedto containing a Thr or Ser at the end of the 2nd β-strand of theRossmanoid fold that helps stability of reaction intermediates;additionally but not limited to containing a Lys at the N-terminus ofthe α-helix C-terminal to the 3rd β-strand of the Rossmanoid fold thathelps stability of reaction intermediates; and additionally but notlimited to containing a GDxxxD, GDxxxxD, DD, or ED signature at the endof the 4th β-strand of the Rossmanoid fold for coordinating magnesium.These features are known in the art and are referenced in, for example,Burroughs et al., Evolutionary Genomics of the HAD Superfamily:Understanding the Structural Adaptations and Catalytic Diversity in aSuperfamily of Phosphoesterases and Allied Enzymes. J. Mol. Biol. 2006;361; 1003-1034.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of a hexose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

Allose

One embodiment of the invention is a process for preparing allose whichincludes converting fructose 6-phosphate (F6P) to psicose 6-phosphate(P6P) catalyzed by psicose 6-phosphate 3-epimerase (P6PE), convertingP6P to allose 6-phosphate (A6P) catalyzed by allose 6-phosphateisomerase (A6PI), and converting the A6P produced to allose catalyzed byallose 6-phosphate phosphatase.

Examples of P6PEs include, but are not limited to the followingproteins, identified by UNIPROT ID numbers: D9TQJ4, A0A0901XZ8, andP32719. Of these, D9TQJ4 and A0A0901XZ8 are obtained from thermophilicorganisms. P32719 is obtained from a mesophilic organism. P32719 is 53%identical to A0A0901XZ8 and 55% identical to D9TQJ4, and each proteincatalyzes the epimerization of F6P to A6P. Furthermore, A0A0901XZ8 is45% identical to D9TQJ4. Conversely, other epimerase proteins identifiedby UNIPROT ID numbers: A0A101D823, R1AXD6, A0A150LBU8, A0A023CQG9, andH1XWY2, which have a degree of identity to D9TQJ4 of 45% or less do notcatalyze the epimerization of F6P to A6P. Examples of P6PEs also includeany homologues having at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% amino acid sequenceidentity to any of the aforementioned Uniprot IDs.

Examples of A6PIs include, but are not limited to Uniprot ID W4V2C8,with the amino acid sequence set forth in SEQ ID NO: 1; and Uniprot IDQ67LX4, with the amino acid sequence set forth in SEQ ID NO: 2. UniprotIDs W4V2C8 and Q67LX4 both catalyze the A6PI reaction and share 56%amino acid sequence identity. Therefore, examples of A6PIs also includeany homologues having at least 55%, preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%, atleast 91%, at least 92%, at least 93%, or at least 94%, and even mostpreferably at least 96, 97, 98, 99 or 100% amino acid sequence identityto any of the aforementioned Uniprot IDs.

A6PIs suitable for use in the process to convert P6P to A6P contain aRossmanoid fold. A mesophilic A6PI described in the art (Mowbray et al.,D-Ribose-5-Phosphate Isomerase B from Escherichia coli is Also aFunctional D-Allose-6-phosphate Isomerase, While the Mycobacteriumtuberculosis Enzyme is Not. J. Mol. Biol. 2008; 382; 667-679) sharesconserved residues with the thermophilic A6PI disclosed in theinvention. In some aspects of the invention the isomerase contains butis not limited to containing a His (mesophilic residue 10) C-terminal tothe 1st β-strand of the Rossmanoid fold for phosphate binding;additionally but not limited to containing an Arg (mesophilic residue133) C-terminal to the α-helix C-terminal to the 5th β-strand of theRossmanoid fold also for phosphate binding; additionally but not limitedto containing a His (mesophilic residue 99) in the active site to ringopen the lactone; additionally but not limited to containing a Cys(mesophilic reside 66) in the active site to act as the catalytic base;additionally but not limited to containing a Thr (mesophilic residue 68)in the active site to act as the catalytic acid; additionally but notlimited to containing a GTG-hydrophobic-G motif near the active site(mesophilic residues 67-71) to stabilize high energy intermediates, andadditionally but not limited to containing a Asn (mesophilic residue100) near the active site to also stabilize high energy intermediates.An A6PI preferably contains all of these conserved residues.

Examples of A6PPs include, but are not limited to the followingproteins: Uniprot ID S9SDA3, with the amino acid sequence set forth inSEQ ID NO: 3; Q9X0Y1, with the amino acid sequence set forth in SEQ IDNO: 4; I3VT81, with the amino acid sequence set forth in SEQ ID NO: 5;A0A132NF06, with the amino acid sequence set forth in SEQ ID NO: 6; andD1C7G9, with the amino acid sequence set forth in SEQ ID NO: 7. UniprotIDs S9SDA3 and I3VT81 both catalyze the A6PP reaction and share 30%amino acid sequence identity. Therefore, examples of A6PPs also includeany homologues having at least 30%, preferably at least 35%, morepreferably at least 40%, more preferably at least 45%, more preferablyat least 50%, more preferably at least 55%, more preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, at least 91%, at least 92%, at least 93%, or at least 94%,and even most preferably at least 96, 97, 98, 99 or 100% amino acidsequence identity to any of the aforementioned Uniprot IDs.

Preferably, an A6PP to convert A6P to allose, contains a Rossmanoid folddomain for catalysis, a Cl capping domain, D×D signature in the 1stβ-strand of the Rossmanoid fold, a Thr or Ser at the end of the 2ndβ-strand of the Rossmanoid fold, a Lys at the N-terminus of the α-helixC-terminal to the 3rd β-strand of the Rossmanoid fold, and a EDsignature at the end of the 4th β-strand of the Rossmanoid fold.

A process for preparing allose according to the invention also includesthe step of enzymatically converting glucose 6-phosphate (G6P) to theF6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In otherembodiments, the process for preparing allose additionally includes thestep of converting glucose 1-phosphate (G1P) to the G6P, where the stepis catalyzed by phosphoglucomutase (PGM). In yet further embodiments,allose production process also includes the step of converting asaccharide to the G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing allose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to P6P via P6PE, (v) converting P6P to A6P via A6P1,and (vi) converting A6P to allose via A6PP. An example of the enzymaticprocess where the saccharide is starch is shown in FIG. 1.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1:1 (αGP:PGM:PGI:P6PE:A6P1:A6PP). To optimize product yields,these ratios can be adjusted in any number of combinations. For example,a ratio of 3:1:1:1:1:1 can be used to maximize the concentration ofphosphorylated intermediates, which will result in increased activity ofthe downstream reactions. Conversely, a ratio of 1:1:1:1:1:3 can be usedto maintain a robust supply of phosphate for αGP, which will result inmore efficient phosphorolytic cleavage of alpha-1,4-glycosidic bonds. Aratio of enzymes, for example, 3:1:1:1:1:3 can be used to furtherincrease the reaction rate. Therefore, the enzyme ratios, includingother optional enzymes discussed below, can be varied to increase theefficiency of allose production. For example, a particular enzyme may bepresent in an amount about 2×, 3×, 4×, 5×, etc. relative to the amountof other enzymes.

Phosphate ions produced by dephosphorylation of A6P can then be recycledin the process step of converting a saccharide to G1P, particularly whenall process steps are conducted in a single bioreactor or reactionvessel. The ability to recycle phosphate in the disclosed processesallows for non-stoichiometric amounts of phosphate to be used, whichkeeps reaction phosphate concentrations low. This affects the overallpathway and the overall rate of the processes, but does not limit theactivity of the individual enzymes and allows for overall efficiency ofthe allose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Low phosphate concentration results in decreased production costs due tolow total phosphate and thus lowered cost of phosphate removal. It alsoprevents inhibition of the A6PP by high concentrations of free phosphateand decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for for making allose involves an energeticallyfavorable reaction.

Allose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to P6P catalyzed byP6PE; converting P6P to A6P catalyzed by A6P1, and converting A6P toallose catalyzed by A6PP. The fructose can be produced, for example, byan enzymatic conversion of sucrose.

Allose can also be produced from sucrose. The process provides an invitro synthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to P6P catalyzed by P6PE;converting P6P to A6P catalyzed by A6P1, and converting A6P to allosecatalyzed by A6PP.

The phosphate ions generated when A6P is converted to allose can then berecycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase allose yields by producing F6Pfrom fructose generated by the phosphorolytic cleavage of sucrose by SP.

In certain embodiments, a process for preparing allose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

Several enzymes can be used to hydrolyze starch to increase the G1Pyield. Such enzymes include isoamylase, pullulanase, and alpha-amylase.Corn starch contains many branches that impede αGP action. Isoamylasecan be used to de-branch starch, yielding linear amylodextrin.Isoamylase-pretreated starch can result in a higher F6P concentration inthe final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to allose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase allose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease allose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to allose through a series of steps. The process provides anin vitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P to P6P catalyzed by P6PE;converting P6P to A6P catalyzed by A6P1, and converting A6P to allosecatalyzed by A6PP. In this process, the phosphate ions can be recycledby the step of converting cellodextrin and cellobiose to G1P.

Several enzymes may be used to hydrolyze solid cellulose towater-soluble cellodextrins and cellobiose. Such enzymes includeendoglucanase and cellobiohydrolase, but not including beta-glucosidase(cellobiase).

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of allose byphosphorylating the degradation product glucose to G6P.

Allose can be produced from glucose. The process involves the steps ofgenerating G6P from glucose and polyphosphate catalyzed by polyphosphateglucokinase (PPGK); converting G6P to F6P catalyzed by PGI; convertingF6P to P6P catalyzed by P6PE; converting P6P to A6P catalyzed by A6P1;and converting A6P to allose catalyzed by A6PP.

Processes of the invention for making allose use low-cost startingmaterials and reduce production costs by decreasing costs associatedwith the feedstock and product separation. Starch, cellulose, sucroseand some of their derivatives are less expensive feedstocks than, forexample, fructose. When allose is produced from psiose, yields are lowerthan in the present invention, and allose must be separated from psicosevia chromatography, which leads to higher production costs.

Also, the step of converting A6P to allose according to the invention isan irreversible phosphatase reaction, regardless of the feedstock.Therefore, allose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of allose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

A particular embodiment of the invention is allose produced by theprocesses described herein for producing allose.

Since allose as similar functionality to sucrose, allose prepared byprocesses of the invention may be added to any beverage or foodstuff toproduce desired sweetness.

Allose prepared by the processes disclosed herein may also be used usedto synergize the effect of potent sweeteners. When combined with one ormore potent sweeteners, allose may be able to effect improvements insensory characteristics such as mouthfeel, flavor and aftertaste of asweetened product. The use of low calorie sweeteners, such as potentsweeteners, in a variety of food products is common place in food andbeverage formulations. For many consumers, however, products marketed asdiet or light versions of products that are artificially sweetened arenot preferred. Attempts have been made over the years to improve thetaste delivery of these diet or light products through the addition ofsmall quantities of carbohydrates. Allose prepared the processes of theinvention would not only able to effect improvements in the quality offood and beverage formulations, particularly in diet/light beverages,but that its use may be synergistic with potent sweeteners such that itis able to replace significant quantities of potent sweeteners, evenwhen it is added at concentrations well below its measured sweet tastethreshold.

Allose produced by processes disclosed herein may be combined with othersweeteners, such as extracts from the Stevia rebaudiana Bertoni plantfor the preparation of low calorie versions of foods such as ice cream.

Allose produced by processes disclosed herein may be used inpresweetened ready to eat (RTE) breakfast cereals and other foodswherein D allose partially or totally replaces sucrose or other commonlyused sugars, as a frosting.

Allose produced by processes disclosed herein may be used as part of asweetener for foods and beverages in combination with sugar alcohols,such as erythritol, and nutritive sweeteners with significant caloriccontent, such as fructose, sucrose, dextrose, maltose, trehalose,rhamnose, corn syrups and fructo-oligosaccharides.

Allose produced by the processes disclosed herein may also be used aspart of a composition that enhances the plant disease control.

Mannose

One embodiment of the invention is a process for preparing mannose whichincludes converting F6P to mannose 6-phosphate (M6P) catalyzed bymannose 6-phosphate isomerase (M6PI); and converting the M6P to mannosecatalyzed by mannose 6-phosphate phosphatase (M6PP).

Examples of M6PIs include, but are not limited to the followingproteins: Uniprot ID A0A1M6TLY7, with the amino acid sequence set forthin SEQ ID NO: 8; H1XQS6, with the amino acid sequence set forth in SEQID NO: 9; G2Q982, with the amino acid sequence set forth in SEQ ID NO:10; and F8F1Z8, with the amino acid sequence set forth in SEQ ID NO: 11.Uniprot IDs G2Q982 and F8F1Z8 both perform the M6PI reaction and share28% amino acid sequence identity. Therefore, examples of M6PIs alsoinclude any homologues having at least 25%, preferably at least 30%,more preferably at least 35%, more preferably at least 40%, morepreferably at least 45%, more preferably at least 50%, more preferablyat least 55%, more preferably at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, at least 91%, atleast 92%, at least 93%, or at least 94%, and even most preferably atleast 96, 97, 98, 99 or 100% amino acid sequence identity to any of theaforementioned Uniprot IDs.

M6PIs suitable for use in the process to convert F6P to M6P contain twodomains with a core of antiparallel β-strands resembling the cupin foldand a third domain consisting of only α-helixes. A M6PI was structurallycharacterized in the art (Sagurthi et al. Structures ofmannose-6-phosphate isomerase from Salmonella typhimurium bound to metalatoms and substrate: implications for catalytic mechanism. Acta Cryst.2009; D65; 724-732) and shares conserved residues with the thermophilicM6PIs described in the invention. In some aspects of the invention theisomerase contains but is not limited to containing a divalent metalcation, preferably Mg²⁺ or Zn²⁺; additionally but not limited tocontaining a Glu and two His residues proposed for use in metal binding(PDB 3H1M residues 134, 99, and 255 respectively); additionally but notlimited to containing an Asp and Lys residue proposed for acid/basecatalysis (PDB 3H1M residues 270 and 132 respectively); and additionallybut not limited to containing a Lys, Pro, and Ala residue proposed forphosphate binding (PDB 3H1M residues 132, 133, and 267 respectively). AnM6PI preferably contains all of these conserved residues.

Examples of M6PPs include, but are not limited to the followingproteins: Uniprot ID A0A1A6DS13, with the amino acid sequence set forthin SEQ ID NO: 12; A0A1M4UN08, with the amino acid sequence set forth inSEQ ID NO: 13; and A0A1N6FCW3, with the amino acid sequence set forth inSEQ ID NO: 14 Uniprot IDs A0A1A6DS13 and A0A1N6FCW3 both catalyze theM6PP reaction and share 35% amino acid sequence identity. Therefore,examples of M6PPs also include any homologues having at least 35%, morepreferably at least 40%, preferably at least 45%, more preferably atleast 50%, more preferably at least 55%, more preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, at least 91%, at least 92%, at least 93%, or at least 94%,and even most preferably at least 96, 97, 98, 99 or 100% amino acidsequence identity to any of the aforementioned Uniprot IDs.

Preferably, an M6PP to convert M6P to mannose contains a Rossmanoid folddomain for catalysis, a C1 capping domain, D×D signature in the 1stβ-strand of the Rossmanoid fold, a Thr or Ser at the end of the 2ndβ-strand of the Rossmanoid fold, a Lys at the N-terminus of the α-helixC-terminal to the 3rd β-strand of the Rossmanoid fold, and a GDxxxDsignature at the end of the 4th β-strand of the Rossmanoid fold.

A process for preparing mannose according to the invention also includesthe step of enzymatically converting glucose 6-phosphate (G6P) to theF6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In otherembodiments, the process for preparing mannose additionally includes thestep of converting glucose 1-phosphate (G1P) to the G6P, where the stepis catalyzed by phosphoglucomutase (PGM). In further embodiments, theprocess for preparing mannose includes the conversion of G6P to F6P toM6P, where this step is catalyzed by bifunctionalphosphoglucose/phosphomannose isomerase (PGPMI). In yet furtherembodiments, mannose production process also includes the step ofconverting a saccharide to the G1P that is catalyzed at least oneenzyme.

Processes of the invention for the production of mannose use PGPMIs thatconvert G6P or F6P to M6P. Examples of PGPMIs include, but are notlimited to the following proteins: Uniprot ID D7CPH7, with the aminoacid sequence set forth in SEQ ID NO: 15; A0A085L170, with the aminoacid sequence set forth in SEQ ID NO: 16; and M1E6Z3, with the aminoacid sequence set forth in SEQ ID NO: 17. Uniprot IDs A0A085L170 andM1E6Z3 both catalyze the PGPMI reaction and share 28% amino acidsequence identity. Therefore, examples of PGPMIs also include anyhomologues having at least 25%, preferably at least 30%, more preferablyat least 35%, more preferably at least 40%, more preferably at least45%, more preferably at least 50%, more preferably at least 55%, morepreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, at least 91%, at least 92%, at least 93%,or at least 94%, and even most preferably at least 96, 97, 98, 99 or100% amino acid sequence identity to any of the aforementioned UniprotIDs.

PGPMI suitable for use in the process to convert G6P or F6P to M6Pcontain two Rossmanoid folds. A PGPMI was structurally characterized inthe art (Swan et al. A Novel Phosphoglucose Isomerase(PGI)/Phosphomannose Isomerase from the Crenarchaeon Pyrobaculumaerophilum Is a Member of the PGI Superfamily. J. Biol. Chem. 2004: 279;39838-39845) and shares conserved residues with the thermophilic PGPMIsdescribed in the invention. In some aspects of the invention theisomerase contains but is not limited to containing a GGS motif (PDB1TZB residues 46-48) where the Gly residues assist in substrate bindingand the Ser residue binds phosphate; additionally but not limited tocontaining a SYSG-X-T-X-ET-Hydrophobic motif (PDB 1TZB residues 87-96)that binds phosphate; additionally but not limited to containing an Argresidue (PDB 1TZB residue 135) that stabilizes high energy intermediatesduring catalysis; additionally but not limited to containing an ENsignature (PDB 1TZB residues 203-204) where the Glu is essential foractive-site base proton transfer; additionally but not limited tocontaining an HN signature (PDB 1TZB residues 219-220) where the His isimportant for ring opening/closure of the substrate during catalysis;and additionally but not limited to containing a conserved Lys residue(PDB 1TZB residue 298) that is important for ring opening/closure of thesubstrate during catalysis. The conserved residues' functions areverified in a separate publication (Hansen et al. BifunctionalPhosphoglucose/Phosphomannose Isomerases from the Archaea Aeropyrumpernix and Thermoplasma acidophilum Constitute a Novel Enzyme Familywithin the Phosphoglucose Isomerase Superfamily. J Biol. Chem. 2004;279; 2262-2272). An PGPMI preferably contains all of these conservedresidues.

Therefore, a process for preparing mannose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to M6P via mannose 6-phosphate isomerase (M6P1, EC5.3.1.8), (v) converting G6P to M6P via bifunctionalphosphoglucose/phosphomannose isomerase (PGPMI, EC 5.3.1.8 and 5.3.1.9),and (vi) converting M6P to mannose via M6PP. An example of the processwhere the saccharide is starch is shown in FIG. 2.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1 (αGP:PGM:PGI:M6P1:M6PP) or 1:1:1:1 (αGP:PGM:PGPMI:M6PP). Tooptimize product yields, these ratios can be adjusted in any number ofcombinations. For example, a ratio of 3:1:1:1:1 can be used to maximizethe concentration of phosphorylated intermediates, which will result inincreased activity of the downstream reactions. Conversely, a ratio of1:1:1:1:3 can be used to maintain a robust supply of phosphate for αGP,which will result in more efficient phosphorolytic cleavage ofalpha-1,4-glycosidic bonds. A ratio of enzymes, for example, 3:1:1:1:3can be used to further increase the reaction rate. Therefore, the enzymeratios, including other optional enzymes discussed below, can be variedto increase the efficiency of mannose production. For example, aparticular enzyme may be present in an amount about 2×, 3×, 4×, 5×, etc.relative to the amount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in a single bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of M6P can then be recycledin the process step of converting a saccharide to G1P, particularly whenall process steps are conducted in a single bioreactor or reactionvessel. The ability to recycle phosphate in the disclosed processesallows for non-stoichiometric amounts of phosphate to be used, whichkeeps reaction phosphate concentrations low. This affects the overallpathway and the overall rate of the processes, but does not limit theactivity of the individual enzymes and allows for overall efficiency ofthe mannose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the M6PP by high concentrationsof free phosphate and decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making mannose involves an energeticallyfavorable reaction.

Mannose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to M6P catalyzed byM6P1; and converting M6P to mannose catalyzed by M6PP. The fructose canbe produced, for example, by an enzymatic conversion of sucrose.

Mannose can also be produced from sucrose. The process provides an invitro synthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to M6P catalyzed by M6P1;and converting M6P to mannose catalyzed by M6PP. In the above steps, theconversion of G6P to F6P to M6P can alternatively be catalyzed by PGPMI.

The phosphate ions generated when M6P is converted to mannose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase mannose yields by producingF6P from fructose generated by the phosphorolytic cleavage of sucrose bySP.

In some embodiments, a process for preparing mannose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to mannose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products tomannose using cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to mannose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase mannose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease mannose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to mannose through a series of steps. The process provides anin vitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P to M6P catalyzed by M6P1; andconverting M6P to mannose catalyzed by M6PP. Alternatively, in theprevious pathway the conversion of G6P to F6P to M6P can be catalyzed byPGPMI. In this process, the phosphate ions can be recycled by the stepof converting cellodextrin and cellobiose to G1P.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of mannose byphosphorylating the degradation product glucose to G6P.

In other embodiments, mannose can be generated from glucose. The processinvolves the steps of generating G6P from glucose and polyphosphatecatalyzed by polyphosphate glucokinase (PPGK); converting G6P to F6Pcatalyzed by PGI; converting F6P to M6P catalyzed by M6P1; andconverting M6P to mannose catalyzed by M6PP. Alternatively, theconversion of G6P to F6P to M6P can be catalyzed by PGPMI.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, fructose.When mannose is produced from fructose, yields are lower than in thepresent invention, and mannose must be separated from fructose viachromatography, which leads to higher production costs.

Also, the step of converting M6P to mannose according to the inventionis an irreversible phosphatase reaction, regardless of the feedstock.Therefore, mannose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of mannose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

A particular embodiment of the invention is mannose produced by theprocesses described herein for producing mannose.

Mannose produced by processes described herein may be used, as discussedabove, in a variety of applications in the pharmaceutical, cosmetic,beverage, food product, dairy, confectionery, and livestock industries.

Additionally, mannose produced by the processes disclosed herein may beconverted to mannitol through hydrogenation. The catalytic hydrogenationof mannose occurs with a stoichiometric yield and gives mannitol. U.S.Pat. No. 5,466,795. Mannitol is widely used in the manufacture ofsugar-free chewing gum, sweets and pharmaceutical excipients. However,the production of high-purity mannose is extremely difficult to achieveand is costly. Id. Accordingly, mannose produced by the aforementionedprocesses can be converted to mannitol via catalytic hydrogenation.

Galactose

One embodiment of the invention is a process for preparing galactosewhich includes converting fructose 6-phosphate (F6P) to tagatose6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE),converting T6P to galactose 6-phosphate (Gal6P) catalyzed by galactose6-phosphate isomerase (Gal6PI), and converting the Gal6P produced togalactose catalyzed by galactose 6-phosphate phosphatase (Gal6PP).

Examples of F6PEs include, but are not limited to the followingproteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. UniprotIDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27%amino acid sequence identity. Therefore, examples of F6PEs also includeany homologues having at least 25%, preferably at least 30%, morepreferably at least 35%, more preferably at least 40%, more preferablyat least 45%, more preferably at least 50%, more preferably at least55%, more preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, at least 91%, at least 92%, atleast 93%, or at least 94%, and even most preferably at least 96, 97,98, 99 or 100% amino acid sequence identity to any of the aforementionedUniprot IDs.

Gal6PI exists as a multimer of two subunits, LacA and LacB. Examples ofGal6PIs include, but are not limited to the following protein(LacA/LacB) subunit pair: Uniprot ID P23494/P23495, with the amino acidsequences set forth in SEQ ID NO: 18/SEQ ID NO: 19. Examples of Gal6PIsalso include any homologues having at least 25%, preferably at least30%, more preferably at least 35%, more preferably at least 40%, morepreferably at least 45%, more preferably at least 50%, more preferablyat least 55%, more preferably at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, at least 91%, atleast 92%, at least 93%, or at least 94%, and even most preferably atleast 96, 97, 98, 99 or 100% amino acid sequence identity to theaforementioned Uniprot ID for LacA subunit and homologues having atleast 25%, at least 30%, more preferably at least 35%, more preferablyat least 40%, more preferably at least 45%, more preferably at least50%, more preferably at least 55%, more preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%, atleast 91%, at least 92%, at least 93%, or at least 94%, and even mostpreferably at least 96, 97, 98, 99 or 100% amino acid sequence identityto the aforementioned Uniprot ID for LacB subunit.

Gal6PIs suitable for use in the process to convert T6P to Gal6P containa heterodimer (‘A’ and ‘B’) consisting of subunits with Rossmann-likeαβα sandwich folds. Conserved residues are discussed in the art (Jung etal. Crystal Structure and Substrate Specificity ofD-Galactose-6-Phosphate Isomerase Complexed with Substrates. PLOS ONE.2013; 8; e72902). In some aspects of the invention the isomeraseheterodimer contains but is not limited to containing Arg130 and Arg134in ‘A’ and His9 and Arg39 in ‘B’ to bind the substrate's phosphategroup; additionally but not limited to containing His96 in ‘A’ for ringopening of substrate; additionally but not limited to containing Asn97in ‘A’ to stabilize high energy intermediates; and additionally butlimited to containing Cys65 and Thr67 of ‘B’ to participate in protontransfer.

Examples of Gal6PPs include, but are not limited to Uniprot ID Q8A2F3with the amino acid sequence set forth in SEQ ID NO: 20. Examples ofGal6PPs also include any homologues having at least 25%, at least 30%,more preferably at least 35%, more preferably at least 40%, morepreferably at least 45%, more preferably at least 50%, more preferablyat least 55%, more preferably at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, at least 91%, atleast 92%, at least 93%, or at least 94%, and even most preferably atleast 96, 97, 98, 99 or 100% amino acid sequence identity to theaforementioned Uniprot ID.

Preferably, a Gal6PP to convert Gal6P to galactose contains a Rossmanoidfold domain for catalysis, a C2 capping domain, D×D signature in the 1stβ-strand of the Rossmanoid fold, a Thr or Ser at the end of the 2ndβ-strand of the Rossmanoid fold, and a GDxxxD signature at the end ofthe 4th β-strand of the Rossmanoid fold.

A process for preparing galactose according to the invention alsoincludes the step of enzymatically converting glucose 6-phosphate (G6P)to the F6P, and this step is catalyzed by phosphoglucoisomerase (PGI).In other embodiments, the process for preparing galactose additionallyincludes the step of converting glucose 1-phosphate (G1P) to the G6P,where the step is catalyzed by phosphoglucomutase (PGM). In yet furtherembodiments, galactose production process also includes the step ofconverting a saccharide to the G1P that is catalyzed at least oneenzyme.

Therefore, a process for preparing galactose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to T6P via F6PE, (v) converting T6P to Gal6P viaGal6PI (EC 5.3.1.26), and (vi) converting Gal6P to galactose via Gal6PP.An example of the process where the saccharide is starch is shown inFIG. 3.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:Gal6PI:Gal6PP). To optimize productyields, these ratios can be adjusted in any number of combinations. Forexample, a ratio of 3:1:1:1:1:1 can be used to maximize theconcentration of phosphorylated intermediates, which will result inincreased activity of the downstream reactions. Conversely, a ratio of1:1:1:1:1:3 can be used to maintain a robust supply of phosphate forαGP, which will result in more efficient phosphorolytic cleavage ofalpha-1,4-glycosidic bonds. A ratio of enzymes, for example, 3:1:1:1:1:3can be used to further increase the reaction rate. Therefore, the enzymeratios, including other optional enzymes discussed below, can be variedto increase the efficiency of galactose production. For example, aparticular enzyme may be present in an amount about 2×, 3×, 4×, 5×, etc.relative to the amount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in a single bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of Gal6P can then berecycled in the process step of converting a saccharide to G1P,particularly when all process steps are conducted in a single bioreactoror reaction vessel. The ability to recycle phosphate in the disclosedprocesses allows for non-stoichiometric amounts of phosphate to be used,which keeps reaction phosphate concentrations low. This affects theoverall pathway and the overall rate of the processes, but does notlimit the activity of the individual enzymes and allows for overallefficiency of the galactose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the Gal6PP by highconcentrations of free phosphate and decreases the potential forphosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making galactose involves an energeticallyfavorable reaction.

Galactose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to T6P catalyzed byF6PE; converting T6P to Gal6P catalyzed by Gal6PI, and converting Gal6Pto galactose catalyzed by Gal6PP. The fructose can be produced, forexample, by an enzymatic conversion of sucrose.

Galactose can be produced from sucrose. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;converting T6P to Gal6P catalyzed by Gal6PI, and converting Gal6P togalactose catalyzed by Gal6PP.

The phosphate ions generated when Gal6P is converted to galactose canthen be recycled in the step of converting sucrose to G1P. Additionally,PPFK and polyphosphate can be used to increase galactose yields byproducing F6P from fructose generated by the phosphorolytic cleavage ofsucrose by SP.

In some embodiments, a process for preparing galactose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to galactose. In certainembodiments, artificial (non-natural) ATP-free enzymatic pathways areprovided to convert starch, cellulose, sucrose, and their derivedproducts to galactose using cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to galactose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase galactose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease galactose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to galactose through a series of steps. The process providesan in vitro synthetic pathway that involves the following steps:generating G1P from cellodextrin and cellobiose and free phosphatecatalyzed by cellodextrin phosphorylase (CDP) and cellobiosephosphorylase (CBP), respectively; converting G1P to G6P catalyzed byPGM; converting G6P to F6P catalyzed by PGI; converting F6P toT6Pcatalyzed by F6PE; converting T6P to Gal6P catalyzed by Gal6PI, andconverting Gal6P to galactose catalyzed by Gal6PP. In this process, thephosphate ions can be recycled by the step of converting cellodextrinand cellobiose to G1P.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of galactose byphosphorylating the degradation product glucose to G6P.

In other embodiments, galactose can be generated from glucose. Theprocess involves the steps of generating G6P from glucose andpolyphosphate catalyzed by polyphosphate glucokinase (PPGK); convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;converting T6P to Gal6P catalyzed by Gal6PI; and converting Gal6P togalactose catalyzed by Gal6PP.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, lactose.When galactose is produced from biomass or lactose, yields are lowerthan in the present invention, and galactose must be separated fromother sugars via chromatography, which leads to higher production costs.Furthermore, our process is animal-free.

The step of converting Gal6P to galactose according to the invention isan irreversible phosphatase reaction, regardless of the feedstock.Therefore, galactose is produced with a very high yield whileeffectively minimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of galactose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

A particular embodiment of the invention is galactose produced by theprocesses described herein for producing galactose.

Fructose

One embodiment of the invention is a process for preparing fructosewhich includes converting fructose 6-phosphate (F6P) to fructosecatalyzed by fructose 6-phosphate phosphatase (F6PP).

A non-limiting example of an F6PP is Uniprot ID B8CWV3, with the aminoacid sequence set forth in SEQ ID NO: 21. Examples of F6PPs also includeany homologues having at least 25%, at least 30%, more preferably atleast 35%, more preferably at least 40%, more preferably at least 45%,more preferably at least 50%, more preferably at least 55%, morepreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, at least 91%, at least 92%, at least 93%,or at least 94%, and even most preferably at least 96, 97, 98, 99 or100% amino acid sequence identity to the aforementioned Uniprot ID.

Preferably, a F6PP to convert F6P to fructose contains a Rossmanoid folddomain for catalysis, a Cl capping domain, D×D signature in the 1stβ-strand of the Rossmanoid fold, a Thr or Ser at the end of the 2ndβ-strand of the Rossmanoid fold, a Lys at the N-terminus of the α-helixC-terminal to the 3rd β-strand of the Rossmanoid fold, and a EDsignature at the end of the 4th β-strand of the Rossmanoid fold.

A process for preparing fructose according to the invention alsoincludes the step of enzymatically converting glucose 6-phosphate (G6P)to the F6P, and this step is catalyzed by phosphoglucoisomerase (PGI).In other embodiments, the process for preparing fructose additionallyincludes the step of converting glucose 1-phosphate (G1P) to the G6P,where the step is catalyzed by phosphoglucomutase (PGM). In yet furtherembodiments, fructose production process also includes the step ofconverting a saccharide to the G1P that is catalyzed at least oneenzyme.

Therefore, a process for preparing fructose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to fructose using F6PP.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1 (αGP:PGM:PGI:F6PP). To optimize product yields, these ratios canbe adjusted in any number of combinations. For example, a ratio of3:1:1:1 can be used to maximize the concentration of phosphorylatedintermediates, which will result in increased activity of the downstreamreactions. Conversely, a ratio of 1:1:1:3 can be used to maintain arobust supply of phosphate for αGP, which will result in more efficientphosphorolytic cleavage of alpha-1,4-glycosidic bonds. A ratio ofenzymes, for example, 3:1:1:3 can be used to further increase thereaction rate. Therefore, the enzyme ratios, including other optionalenzymes discussed below, can be varied to increase the efficiency offructose production. For example, a particular enzyme may be present inan amount about 2×, 3×, 4×, 5×, etc. relative to the amount of otherenzymes.

One of the important advantages of the processes is that the processsteps can be conducted in a single bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of F6P can then be recycledin the process step of converting a saccharide to G1P, particularly whenall process steps are conducted in a single bioreactor or reactionvessel. The ability to recycle phosphate in the disclosed processesallows for non-stoichiometric amounts of phosphate to be used, whichkeeps reaction phosphate concentrations low. This affects the overallpathway and the overall rate of the processes, but does not limit theactivity of the individual enzymes and allows for overall efficiency ofthe fructose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the F6PP by high concentrationsof free phosphate and decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making fructose involves an energeticallyfavorable reaction.

Fructose can also be produced from sucrose via an F6P intermediate. Theprocess provides an in vitro synthetic pathway that includes thefollowing enzymatic steps: generating G1P from sucrose and freephosphate catalyzed by sucrose phosphorylase (SP); converting G1P to G6Pcatalyzed by PGM; converting G6P to F6P catalyzed by PGI; F6P tofructose catalyzed by F6PP. An example enzymatic pathway is provided inFIG. 5

The phosphate ions generated when F6P is converted to fructose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase fructose yields by producingF6P from fructose generated by the phosphorolytic cleavage of sucrose bySP.

In some embodiments, a process for preparing fructose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to fructose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products tofructose using cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to fructose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase fructose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease fructose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to fructose through a series of steps. The process provides anin vitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P to fructose catalyzed by F6PP. Inthis process, the phosphate ions can be recycled by the step ofconverting cellodextrin and cellobiose to G1P.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of fructose byphosphorylating the degradation product glucose to G6P.

In other embodiments, fructose can be generated from glucose. Theprocess involves the steps of generating G6P from glucose andpolyphosphate catalyzed by polyphosphate glucokinase (PPGK); convertingG6P to F6P catalyzed by PGI; converting F6P to fructose catalyzed byF6PP.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, lactose.When fructose is produced from biomass or lactose, yields are lower thanin the present invention, and fructose must be separated from othersugars via chromatography, which leads to higher production costs.Furthermore, our process is animal-free.

The step of converting F6P to fructose according to the invention is anirreversible phosphatase reaction, regardless of the feedstock.Therefore, fructose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of fructose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

Ar particular embodiment of the invention is fructose produced by theprocesses described herein for producing fructose.

Altrose

One embodiment of the invention is a process for preparing altrose whichincludes converting fructose 6-phosphate (F6P) to psicose 6-phosphate(P6P) catalyzed by psicose 6-phosphate 3-epimerase (P6PE), convertingP6P to altrose 6-phosphate (Alt6P) catalyzed by altrose 6-phosphateisomerase (Alt6PI), and converting the Alt6P produced to altrosecatalyzed by altrose 6-phosphate phosphatase.

A process for preparing altrose according to the invention also includesthe step of enzymatically converting glucose 6-phosphate (G6P) to theF6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In otherembodiments, the process for preparing altrose additionally includes thestep of converting glucose 1-phosphate (G1P) to the G6P, where the stepis catalyzed by phosphoglucomutase (PGM). In yet further embodiments,altrose production process also includes the step of converting asaccharide to the G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing altrose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to P6P via P6PE, (v) converting P6P to Alt6P viaAlt6PI, and (vi) converting Alt6P to altrose via Alt6PP. An example ofthe enzymatic process where the saccharide is starch is shown in FIG. 1.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1:1 (αGP:PGM:PGI:P6PE:Alt6PI:Alt6PP). To optimize productyields, these ratios can be adjusted in any number of combinations. Forexample, a ratio of 3:1:1:1:1:1 can be used to maximize theconcentration of phosphorylated intermediates, which will result inincreased activity of the downstream reactions. Conversely, a ratio of1:1:1:1:1:3 can be used to maintain a robust supply of phosphate forαGP, which will result in more efficient phosphorolytic cleavage ofalpha-1,4-glycosidic bonds. A ratio of enzymes, for example, 3:1:1:1:1:3can be used to further increase the reaction rate. Therefore, the enzymeratios, including other optional enzymes discussed below, can be variedto increase the efficiency of altrose production. For example, aparticular enzyme may be present in an amount about 2×, 3×, 4×, 5×, etc.relative to the amount of other enzymes.

Phosphate ions produced by dephosphorylation of Alt6P can then berecycled in the process step of converting a saccharide to G1P,particularly when all process steps are conducted in a single bioreactoror reaction vessel. The ability to recycle phosphate in the disclosedprocesses allows for non-stoichiometric amounts of phosphate to be used,which keeps reaction phosphate concentrations low. This affects theoverall pathway and the overall rate of the processes, but does notlimit the activity of the individual enzymes and allows for overallefficiency of the altrose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concentration results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the Alt6PP by highconcentrations of free phosphate and decreases the potential forphosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making altrose involves an energeticallyfavorable reaction.

Altrose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to P6P catalyzed byP6PE; converting P6P to Alt6P catalyzed by Alt6PI, and converting Alt6Pto altrose catalyzed by Alt6PP. The fructose can be produced, forexample, by an enzymatic conversion of sucrose.

Altrose can also be produced from sucrose. The process provides an invitro synthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to P6P catalyzed by P6PE;converting P6P to Alt6P catalyzed by Alt6PI, and converting Alt6P toaltrose catalyzed by Alt6PP.

The phosphate ions generated when Alt6P is converted to altrose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase altrose yields by producingF6P from fructose generated by the phosphorolytic cleavage of sucrose bySP.

In certain embodiments, a process for preparing altrose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

Several enzymes can be used to hydrolyze starch to increase the G1Pyield. Such enzymes include isoamylase, pullulanase, and alpha-amylase.Corn starch contains many branches that impede αGP action. Isoamylasecan be used to de-branch starch, yielding linear amylodextrin.Isoamylase-pretreated starch can result in a higher F6P concentration inthe final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to altrose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase altrose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease altrose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to altrose through a series of steps. The process provides anin vitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P to P6P catalyzed by P6PE;converting P6P to Alt6P catalyzed by Alt6PI, and converting Alt6P toaltrose catalyzed by Alt6PP. In this process, the phosphate ions can berecycled by the step of converting cellodextrin and cellobiose to G1P.

Several enzymes may be used to hydrolyze solid cellulose towater-soluble cellodextrins and cellobiose. Such enzymes includeendoglucanase and cellobiohydrolase, but not including beta-glucosidase(cellobiase).

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of altrose byphosphorylating the degradation product glucose to G6P.

Altrose can be produced from glucose. The process involves the steps ofgenerating G6P from glucose and polyphosphate catalyzed by polyphosphateglucokinase (PPGK); converting G6P to F6P catalyzed by PGI; convertingF6P to P6P catalyzed by P6PE; converting P6P to Alt6P catalyzed by A6P1;and converting Alt6P to altrose catalyzed by Alt6PP.

Processes of the invention for making altrose use low-cost startingmaterials and reduce production costs by decreasing costs associatedwith the feedstock and product separation. Starch, cellulose, sucroseand some of their derivatives are less expensive feedstocks than, forexample, fructose. When altrose is produced from psiose, yields arelower than in the present invention, and altrose must be separated frompsicose via chromatography, which leads to higher production costs.

Also, the step of converting Alt6P to altrose according to the inventionis an irreversible phosphatase reaction, regardless of the feedstock.Therefore, altrose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of altrose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

Ar particular embodiment of the invention is altrose produced by theprocesses described herein for producing altrose.

Talose

One embodiment of the invention is a process for preparing talose whichincludes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate(T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), convertingT6P to talose 6-phosphate (Tal6P) catalyzed by talose 6-phosphateisomerase (Tal6PI), and converting the Tal6P produced to talosecatalyzed by talose 6-phosphate phosphatase (Tal6PP).

Examples of F6PEs include, but are not limited to the followingproteins: Uniprot ID E8NON6, E4SEH3, I0I507, H1XRG1, and B5YBD7. UniprotIDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27%amino acid sequence identity. Therefore, examples of F6PEs also includeany homologues having at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% amino acid sequence identity to anyof the aforementioned Uniprot IDs.

A process for preparing talose according to the invention also includesthe step of enzymatically converting glucose 6-phosphate (G6P) to theF6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In otherembodiments, the process for preparing talose additionally includes thestep of converting glucose 1-phosphate (G1P) to the G6P, where the stepis catalyzed by phosphoglucomutase (PGM). In yet further embodiments,talose production process also includes the step of converting asaccharide to the G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing talose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to T6P via F6PE, (v) converting T6P to Tal6P viaTal6PI (EC 5.3.1.26), and (vi) converting Tal6P to talose via Tal6PP. Anexample of the process where the saccharide is starch is shown in FIG.3.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:Tal6PI:Tal6PP). To optimize productyields, these ratios can be adjusted in any number of combinations. Forexample, a ratio of 3:1:1:1:1:1 can be used to maximize theconcentration of phosphorylated intermediates, which will result inincreased activity of the downstream reactions. Conversely, a ratio of1:1:1:1:1:3 can be used to maintain a robust supply of phosphate forαGP, which will result in more efficient phosphorolytic cleavage ofalpha-1,4-glycosidic bonds. A ratio of enzymes, for example, 3:1:1:1:1:3can be used to further increase the reaction rate. Therefore, the enzymeratios, including other optional enzymes discussed below, can be variedto increase the efficiency of talose production. For example, aparticular enzyme may be present in an amount about 2×, 3×, 4×, 5×, etc.relative to the amount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in a single bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of Tal6P can then berecycled in the process step of converting a saccharide to G1P,particularly when all process steps are conducted in a single bioreactoror reaction vessel. The ability to recycle phosphate in the disclosedprocesses allows for non-stoichiometric amounts of phosphate to be used,which keeps reaction phosphate concentrations low. This affects theoverall pathway and the overall rate of the processes, but does notlimit the activity of the individual enzymes and allows for overallefficiency of the talose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the Tal6PP by highconcentrations of free phosphate and decreases the potential forphosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making talose involves an energeticallyfavorable reaction.

Talose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to T6P catalyzed byF6PE; converting T6P to Tal6P catalyzed by Tal6PI, and converting Tal6Pto talose catalyzed by Tal6PP. The fructose can be produced, forexample, by an enzymatic conversion of sucrose.

Talose can be produced from sucrose. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;converting T6P to Tal6P catalyzed by Tal6PI, and converting Tal6P totalose catalyzed by Tal6PP.

The phosphate ions generated when Tal6P is converted to talose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase talose yields by producing F6Pfrom fructose generated by the phosphorolytic cleavage of sucrose by SP.

In some embodiments, a process for preparing talose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to talose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products to taloseusing cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to talose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase talose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease talose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to talose through a series of steps. The process provides anin vitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;converting T6P to Tal6P catalyzed by Tal6PI, and converting Tal6P totalose catalyzed by Tal6PP. In this process, the phosphate ions can berecycled by the step of converting cellodextrin and cellobiose to G1P.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of talose byphosphorylating the degradation product glucose to G6P.

In other embodiments, talose can be generated from glucose. The processinvolves the steps of generating G6P from glucose and polyphosphatecatalyzed by polyphosphate glucokinase (PPGK); converting G6P to F6Pcatalyzed by PGI; converting F6P to T6P catalyzed by F6PE; convertingT6P to Tal6P catalyzed by Tal6PI; and converting Tal6P to talosecatalyzed by Tal6PP.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, lactose.When talose is produced from biomass or lactose, yields are lower thanin the present invention, and talose must be separated from other sugarsvia chromatography, which leads to higher production costs. Furthermore,our process is animal-free.

The step of converting Tal6P to talose according to the invention is anirreversible phosphatase reaction, regardless of the feedstock.Therefore, talose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of talose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

A particular embodiment of the invention is talose produced by theprocesses described herein for producing talose.

Sorbose

One embodiment of the invention is a process for preparing sorbose whichincludes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate(T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), convertingT6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphateepimerase (S6PE), and converting the S6P produced to sorbose catalyzedby sorbose 6-phosphate phosphatase (S6PP).

Examples of F6PEs include, but are not limited to the followingproteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. UniprotIDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27%amino acid sequence identity. Therefore, examples of F6PEs also includeany homologues having at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% amino acid sequence identity to anyof the aforementioned Uniprot IDs.

A process for preparing sorbose according to the invention also includesthe step of enzymatically converting glucose 6-phosphate (G6P) to theF6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In otherembodiments, the process for preparing sorbose additionally includes thestep of converting glucose 1-phosphate (G1P) to the G6P, where the stepis catalyzed by phosphoglucomutase (PGM). In yet further embodiments,sorbose production process also includes the step of converting asaccharide to the G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing sorbose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to T6P via F6PE, (v) converting T6P to S6P via S6PE(EC 5.3.1.26), and (vi) converting S6P to sorbose via S6PP. An exampleof the process where the saccharide is starch is shown in FIG. 3.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:S6PE:S6PP). To optimize product yields,these ratios can be adjusted in any number of combinations. For example,a ratio of 3:1:1:1:1:1 can be used to maximize the concentration ofphosphorylated intermediates, which will result in increased activity ofthe downstream reactions. Conversely, a ratio of 1:1:1:1:1:3 can be usedto maintain a robust supply of phosphate for αGP, which will result inmore efficient phosphorolytic cleavage of alpha-1,4-glycosidic bonds. Aratio of enzymes, for example, 3:1:1:1:1:3 can be used to furtherincrease the reaction rate. Therefore, the enzyme ratios, includingother optional enzymes discussed below, can be varied to increase theefficiency of sorbose production. For example, a particular enzyme maybe present in an amount about 2×, 3×, 4×, 5×, etc. relative to theamount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in a single bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of S6P can then be recycledin the process step of converting a saccharide to G1P, particularly whenall process steps are conducted in a single bioreactor or reactionvessel. The ability to recycle phosphate in the disclosed processesallows for non-stoichiometric amounts of phosphate to be used, whichkeeps reaction phosphate concentrations low. This affects the overallpathway and the overall rate of the processes, but does not limit theactivity of the individual enzymes and allows for overall efficiency ofthe sorbose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the S6PP by high concentrationsof free phosphate and decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making sorbose involves an energeticallyfavorable reaction.

Sorbose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to T6P catalyzed byF6PE; converting T6P to S6P catalyzed by S6PE, and converting S6P tosorbose catalyzed by S6PP. The fructose can be produced, for example, byan enzymatic conversion of sucrose.

Sorbose can be produced from sucrose. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;converting T6P to S6P catalyzed by S6PE, and converting S6P to sorbosecatalyzed by S6PP.

The phosphate ions generated when S6P is converted to sorbose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase sorbose yields by producingF6P from fructose generated by the phosphorolytic cleavage of sucrose bySP.

In some embodiments, a process for preparing sorbose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to sorbose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products tosorbose using cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to sorbose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase sorbose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease sorbose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to sorbose through a series of steps. The process provides anin vitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P toT6P catalyzed by F6PE; convertingT6P to S6P catalyzed by S6PE, and converting S6P to sorbose catalyzed byS6PP. In this process, the phosphate ions can be recycled by the step ofconverting cellodextrin and cellobiose to G1P.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of sorbose byphosphorylating the degradation product glucose to G6P.

In other embodiments, sorbose can be generated from glucose. The processinvolves the steps of generating G6P from glucose and polyphosphatecatalyzed by polyphosphate glucokinase (PPGK); converting G6P to F6Pcatalyzed by PGI; converting F6P to T6P catalyzed by F6PE; convertingT6P to S6P catalyzed by S6PE; and converting S6P to sorbose catalyzed byS6PP.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, lactose.When sorbose is produced from biomass or lactose, yields are lower thanin the present invention, and sorbose must be separated from othersugars via chromatography, which leads to higher production costs.Furthermore, our process is animal-free.

The step of converting S6P to sorbose according to the invention is anirreversible phosphatase reaction, regardless of the feedstock.Therefore, sorbose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of sorbose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

A particular embodiment of the invention is sorbose produced by theprocesses described herein for producing sorbose.

Gulose

One embodiment of the invention is a process for preparing gulose whichincludes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate(T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), convertingT6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphateepimerase (S6PE), converting the S6P produced to gulose 6-phosphate(Gul6P) catalyzed by gulose 6-phosphate isomerase and converting theGul6P to gulose by gulose 6-phosphate phosphatase (Gul6PP).

Examples of F6PEs include, but are not limited to the followingproteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. UniprotIDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27%amino acid sequence identity. Therefore, examples of F6PEs also includeany homologues having at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% amino acid sequence identity to anyof the aforementioned Uniprot IDs.

A process for preparing gulose according to the invention also includesthe step of enzymatically converting glucose 6-phosphate (G6P) to theF6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In otherembodiments, the process for preparing gulose additionally includes thestep of converting glucose 1-phosphate (G1P) to the G6P, where the stepis catalyzed by phosphoglucomutase (PGM). In yet further embodiments,gulose production process also includes the step of converting asaccharide to the G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing gulose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to T6P via F6PE, (v) converting T6P to S6P via S6PE(EC 5.3.1.26), (vi) converting S6P to Gul6P via Gul6PI, and (vii)converting GuIP to gulose via Gul6PP.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:S6PE:Gul6PI:GuIPP). To optimize productyields, these ratios can be adjusted in any number of combinations. Forexample, a ratio of 3:1:1:1:1:1:1 can be used to maximize theconcentration of phosphorylated intermediates, which will result inincreased activity of the downstream reactions. Conversely, a ratio of1:1:1:1:1:1:3 can be used to maintain a robust supply of phosphate forαGP, which will result in more efficient phosphorolytic cleavage ofalpha-1,4-glycosidic bonds. A ratio of enzymes, for example,3:1:1:1:1:1:3 can be used to further increase the reaction rate.Therefore, the enzyme ratios, including other optional enzymes discussedbelow, can be varied to increase the efficiency of gulose production.For example, a particular enzyme may be present in an amount about 2×,3×, 4×, 5×, etc. relative to the amount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in a single bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of S6P can then be recycledin the process step of converting a saccharide to G1P, particularly whenall process steps are conducted in a single bioreactor or reactionvessel. The ability to recycle phosphate in the disclosed processesallows for non-stoichiometric amounts of phosphate to be used, whichkeeps reaction phosphate concentrations low. This affects the overallpathway and the overall rate of the processes, but does not limit theactivity of the individual enzymes and allows for overall efficiency ofthe gulose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the S6PP by high concentrationsof free phosphate and decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making gulose involves an energeticallyfavorable reaction.

Gulose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to T6P catalyzed byF6PE; converting T6P to S6P catalyzed by S6PE, S6P to Gul6P by Gul6PI,and Gul6P to gulose by Gul6PP. The fructose can be produced, forexample, by an enzymatic conversion of sucrose.

Gulose can be produced from sucrose. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;converting T6P to S6P catalyzed by S6PE, S6P to Gul6P by Gul6PI, andGul6P to gulose by Gul6PP.

The phosphate ions generated when S6P is converted to sorbose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase gulose yields by producing F6Pfrom fructose generated by the phosphorolytic cleavage of sucrose by SP.

In some embodiments, a process for preparing gulose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to gulose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products to guloseusing cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to gulose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase gulose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease gulose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to gulose through a series of steps. The process provides anin vitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P toT6P catalyzed by F6PE; convertingT6P to S6P catalyzed by S6PE, and converting S6P to sorbose catalyzed byS6PP. In this process, the phosphate ions can be recycled by the step ofconverting cellodextrin and cellobiose to G1P.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of gulose byphosphorylating the degradation product glucose to G6P.

In other embodiments, gulose can be generated from glucose. The processinvolves the steps of generating G6P from glucose and polyphosphatecatalyzed by polyphosphate glucokinase (PPGK); converting G6P to F6Pcatalyzed by PGI; converting F6P to T6P catalyzed by F6PE; convertingT6P to S6P catalyzed by S6PE; S6P to Gul6P by Gul6PI, and Gul6P togulose by Gul6PP.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, lactose.When gulose is produced from biomass or lactose, yields are lower thanin the present invention, and gulose must be separated from other sugarsvia chromatography, which leads to higher production costs. Furthermore,our process is animal-free.

The step of converting S6P to gulose according to the invention is anirreversible phosphatase reaction, regardless of the feedstock.Therefore, gulose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of gulose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

A particular embodiment of the invention is gulose produced by theprocesses described herein for producing gulose.

Idose

One embodiment of the invention is a process for preparing idose whichincludes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate(T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), convertingT6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphateepimerase (S6PE), converting the S6P produced to idose 6-phosphate (16P)catalyzed by idose 6-phosphate isomerase and converting the 16P to idoseby idose 6-phosphate phosphatase (16PP).

Examples of F6PEs include, but are not limited to the followingproteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. UniprotIDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27%amino acid sequence identity. Therefore, examples of F6PEs also includeany homologues having at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% amino acid sequence identity to anyof the aforementioned Uniprot IDs.

A process for preparing idose according to the invention also includesthe step of enzymatically converting glucose 6-phosphate (G6P) to theF6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In otherembodiments, the process for preparing idose additionally includes thestep of converting glucose 1-phosphate (G1P) to the G6P, where the stepis catalyzed by phosphoglucomutase (PGM). In yet further embodiments,idose production process also includes the step of converting asaccharide to the G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing idose according to the invention can,for example, include the following steps: (i) converting a saccharide toglucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1Pto G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6Pto F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convertingF6P to T6P via F6PE, (v) converting T6P to S6P via S6PE (EC 5.3.1.26),(vi) converting S6P to 16P via 16P1, and (vii) converting 16P to idosevia 16PP.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:S6PE:16P1:16PP). To optimize productyields, these ratios can be adjusted in any number of combinations. Forexample, a ratio of 3:1:1:1:1:1:1 can be used to maximize theconcentration of phosphorylated intermediates, which will result inincreased activity of the downstream reactions. Conversely, a ratio of1:1:1:1:1:1:3 can be used to maintain a robust supply of phosphate forαGP, which will result in more efficient phosphorolytic cleavage ofalpha-1,4-glycosidic bonds. A ratio of enzymes, for example,3:1:1:1:1:1:3 can be used to further increase the reaction rate.Therefore, the enzyme ratios, including other optional enzymes discussedbelow, can be varied to increase the efficiency of idose production. Forexample, a particular enzyme may be present in an amount about 2×, 3×,4×, 5×, etc. relative to the amount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in a single bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of S6P can then be recycledin the process step of converting a saccharide to G1P, particularly whenall process steps are conducted in a single bioreactor or reactionvessel. The ability to recycle phosphate in the disclosed processesallows for non-stoichiometric amounts of phosphate to be used, whichkeeps reaction phosphate concentrations low. This affects the overallpathway and the overall rate of the processes, but does not limit theactivity of the individual enzymes and allows for overall efficiency ofthe idose making processes.

For example, reaction phosphate concentrations can range from about 0.1mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mMto about 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the S6PP by high concentrationsof free phosphate and decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(P)(H), i.e., NAD(P)(H)-free.Other advantages also include the fact that at least one step of thedisclosed processes for making idose involves an energetically favorablereaction.

Idose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to T6P catalyzed byF6PE; converting T6P to S6P catalyzed by S6PE, S6P to 16P by 16P1, and16P to idose by 16PP. The fructose can be produced, for example, by anenzymatic conversion of sucrose.

Idose can be produced from sucrose. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;converting T6P to S6P catalyzed by S6PE, S6P to 16P by 16PI, and 16P toidose by 16PP.

The phosphate ions generated when S6P is converted to sorbose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase idose yields by producing F6Pfrom fructose generated by the phosphorolytic cleavage of sucrose by SP.

In some embodiments, a process for preparing idose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to F6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to idose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products to idoseusing cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to idose and increasedsolubility.

Maltose phosphorylase (MP) can be used to increase idose yields byphosphorolytically cleaving the degradation product maltose into G1P andglucose. Alternatively, 4-glucan transferase (4GT) can be used toincrease idose yields by recycling the degradation products glucose,maltose, and maltotriose into longer maltooligosaccharides; which can bephosphorolytically cleaved by αGP to yield G1P.

In certain embodiments, cellulose and its derived products can beconverted to idose through a series of steps. The process provides an invitro synthetic pathway that involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P toT6P catalyzed by F6PE; convertingT6P to S6P catalyzed by S6PE, and converting S6P to sorbose catalyzed byS6PP. In this process, the phosphate ions can be recycled by the step ofconverting cellodextrin and cellobiose to G1P.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of idose byphosphorylating the degradation product glucose to G6P.

In other embodiments, idose can be generated from glucose. The processinvolves the steps of generating G6P from glucose and polyphosphatecatalyzed by polyphosphate glucokinase (PPGK); converting G6P to F6Pcatalyzed by PGI; converting F6P to T6P catalyzed by F6PE; convertingT6P to S6P catalyzed by S6PE; S6P to 16P by 16P1, and 16P to idose by16PP.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, lactose.When idose is produced from biomass or lactose, yields are lower than inthe present invention, and idose must be separated from other sugars viachromatography, which leads to higher production costs. Furthermore, ourprocess is animal-free.

The step of converting S6P to idose according to the invention is anirreversible phosphatase reaction, regardless of the feedstock.Therefore, idose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of idose, has relatively high reaction rates dueto the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

A particular embodiment of the invention is idose produced by theprocesses described herein for producing idose.

Tagatose

Processes for making tagatose include converting F6P to T6P, catalyzedby an epimerase; and converting the T6P to tagatose, catalyzed by aphosphatase.

Epimerases suitable for use in the processes to convert F6P to T6Pinclude F6PEs. Examples of F6PEs include, but are not limited to thefollowing proteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, andB5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reactionand share 27% amino acid sequence identity. Therefore, examples of F6PEsalso include any homologues having at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% amino acid sequenceidentity to any of the aforementioned Uniprot IDs.

Phosphatases that convert T6P to tagatose (D-tagatose), T6PPs may beused in a process. Examples of T6PPs include, but are not limited to thefollowing proteins: Uniprot ID 029805, D2RHV2 and F2KMK2. Uniprot IDs029805 and F2KMK2 both catalyze the F6PE reaction and share 67% aminoacid sequence identity. Therefore, examples of T6PPs also include anyhomologues having at least 65%, preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, 97%, 98%, 99%, or 100% amino acidsequence identity to any of the aforementioned Uniprot IDs.

A process for preparing tagatose also includes the step of enzymaticallyconverting glucose 6-phosphate (G6P) to the F6P, and this step iscatalyzed by phosphoglucose isomerase (PGI). The process for preparingtagatose additionally includes the step of converting glucose1-phosphate (G1P) to the G6P, where the step is catalyzed byphosphoglucomutase (PGM). Furthermore, tagatose production process alsoincludes the step of converting a saccharide to the G1P that iscatalyzed at least one enzyme.

Therefore, a process for preparing tagatose, for example, include thefollowing steps: (i) converting a saccharide to glucose 1-phosphate(G1P) using one or more enzymes; (ii) converting G1P to G6P usingphosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P usingphosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P viafructose 6-phosphate epimerase (F6PE), and (v) converting T6P totagatose via tagatose 6-phosphate phosphatase (T6PP).

Typically, the ratios of enzyme units used in the process are 1:1:1:1:1(αGP:PGM:PGI:F6PE:T6PP). To optimize product yields, these ratios can beadjusted in any number of combinations. For example, a ratio of3:1:1:1:1 can be used to maximize the concentration of phosphorylatedintermediates, which will result in increased activity of the downstreamreactions. Conversely, a ratio of 1:1:1:1:3 can be used to maintain arobust supply of phosphate for αGP, which will result in more efficientphosphorolytic cleavage of alpha-1,4-glycosidic bonds. A ratio ofenzymes, for example, 3:1:1:1:3 can be used to further increase thereaction rate. Therefore, the enzyme ratios, including other optionalenzymes discussed below, can be varied to increase the efficiency oftagatose production. For example, a particular enzyme may be present inan amount about 2×, 3×, 4×, 5×, etc. relative to the amount of otherenzymes.

Tagatose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to T6P catalyzed byF6PE; and converting T6P to tagatose catalyzed by T6PP. The fructose canbe produced, for example, by an enzymatic conversion of sucrose.

Tagatose can be produced from sucrose. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE;and converting T6P to tagatose catalyzed by T6PP.

The phosphate ions generated when T6P is converted to tagatose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase tagatose yields by producingF6P from fructose generated by the phosphorolytic cleavage of sucrose bySP.

A process for preparing tagatose includes the following steps:generating glucose from polysaccharides and oligosaccharides byenzymatic hydrolysis or acid hydrolysis, converting glucose to G6Pcatalyzed by at least one enzyme, generating fructose frompolysaccharides and oligosaccharides by enzymatic hydrolysis or acidhydrolysis, and converting fructose to G6P catalyzed by at least oneenzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

Cellulose and its derived products can be converted to tagatose througha series of steps. The process involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P to T6P catalyzed by F6PE; andconverting T6P to tagatose catalyzed by T6PP. In this process, thephosphate ions can be recycled by the step of converting cellodextrinand cellobiose to G1P.

Tagatose can be generated from glucose. The process involves the stepsof generating G6P from glucose and polyphosphate catalyzed bypolyphosphate glucokinase (PPGK); converting G6P to F6P catalyzed byPGI; converting F6P to T6P catalyzed by F6PE; and converting T6P totagatose catalyzed by T6PP.

Psicose

Processes for making psicose include converting fructose 6-phosphate(F6P) to psicose 6-phosphate (P6P) catalyzed by an epimerase (e.g.,psicose 6-phosphate 3-epimerase, P6PE) and converting the P6P producedto psicose catalyzed by a phosphatase (e.g., psicose 6-phosphatephosphatase, P6PP).

Examples of P6PEs include, but are not limited to the followingproteins, identified by UNIPROT ID numbers: D9TQJ4, A0A0901XZ8, andP32719. Uniprot IDs A0A0901XZ8 and D9TQJ4 both catalyze the P6PEreaction and share 45% amino acid sequence identity. Therefore, examplesof P6PEs also include any homologues having at least 45%, preferably atleast 50%, more preferably at least 55%, more preferably at least 60%,more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 96, 97, 98, 99 or 100% toany of the aforementioned Uniprot IDs.

Examples of P6PPs include, but are not limited to the followingproteins: Uniprot ID. A3DC21, Q5LGR4, and Q89ZR1. Uniprot IDs A3DC21 andQ89ZR1 both catalyze the P6PP reaction and share 45% amino acid sequenceidentity. Therefore, examples of P6PPs also include any homologueshaving at least 45%, preferably at least 50%, more preferably at least55%, more preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96, 97, 98, 99 or 100% to any of the aforementioned Uniprot IDs.

A process for preparing psicose also includes the step of enzymaticallyconverting glucose 6-phosphate (G6P) to the F6P, and this step iscatalyzed by phosphoglucose isomerase (PGI). The process for preparingpsicose additionally includes the step of converting glucose 1-phosphate(G1P) to the G6P, where the step is catalyzed by phosphoglucomutase(PGM). Furthermore, psicose production process also includes the step ofconverting a saccharide to the G1P that is catalyzed at least oneenzyme.

Therefore, a process for preparing psicose, for example, include thefollowing steps: (i) converting a saccharide to glucose 1-phosphate(G1P) using one or more enzymes; (ii) converting G1P to G6P usingphosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P usingphosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to P6P viapsicose 6-phosphate epimerase (P6PE), and (v) converting P6P to psicosevia psicose 6-phosphate phosphatase (P6PP).

Typically, the ratios of enzyme units used in the process are 1:1:1:1:1(αGP:PGM:PGI:P6PE:P6PP). To optimize product yields, these ratios can beadjusted in any number of combinations. For example, a ratio of3:1:1:1:1 can be used to maximize the concentration of phosphorylatedintermediates, which will result in increased activity of the downstreamreactions. Conversely, a ratio of 1:1:1:1:3 can be used to maintain arobust supply of phosphate for αGP, which will result in more efficientphosphorolytic cleavage of alpha-1,4-glycosidic bonds. A ratio ofenzymes, for example, 3:1:1:1:3 can be used to further increase thereaction rate. Therefore, the enzyme ratios, including other optionalenzymes discussed below, can be varied to increase the efficiency oftagatose production. For example, a particular enzyme may be present inan amount about 2×, 3×, 4×, 5×, etc. relative to the amount of otherenzymes.

Psicose can also be produced from fructose. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to P6P catalyzed byP6PE; and converting P6P to psicose catalyzed by P6PP. The fructose canbe produced, for example, by an enzymatic conversion of sucrose.

Psicose can be produced from sucrose. The process includes the followingenzymatic steps: generating G1P from sucrose and free phosphatecatalyzed by sucrose phosphorylase (SP); converting G1P to G6P catalyzedby PGM; converting G6P to F6P catalyzed by PGI; converting F6P to P6Pcatalyzed by P6PE; and converting P6P to psicose catalyzed by P6PP.

The phosphate ions generated when P6P is converted to psicose can thenbe recycled in the step of converting sucrose to G1P. Additionally, PPFKand polyphosphate can be used to increase psicose yields by producingF6P from fructose generated by the phosphorolytic cleavage of sucrose bySP.

A process for preparing psicose includes the following steps: generatingglucose from polysaccharides and oligosaccharides by enzymatichydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by atleast one enzyme, generating fructose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, andconverting fructose to G6P catalyzed by at least one enzyme. Examples ofthe polysaccharides and oligosaccharides are enumerated above.

Cellulose and its derived products can be converted to psicose through aseries of steps. The process involves the following steps: generatingG1P from cellodextrin and cellobiose and free phosphate catalyzed bycellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP),respectively; converting G1P to G6P catalyzed by PGM; converting G6P toF6P catalyzed by PGI; converting F6P to P6P catalyzed by P6PE; andconverting P6P to psicose catalyzed by P6PP. In this process, thephosphate ions can be recycled by the step of converting cellodextrinand cellobiose to G1P.

Psicose can be generated from glucose. The process involves the steps ofgenerating G6P from glucose and polyphosphate catalyzed by polyphosphateglucokinase (PPGK); converting G6P to F6P catalyzed by PGI; convertingF6P to P6P catalyzed by P6PE; and converting P6P to psicose catalyzed byP6PP.

EXAMPLES

Materials and Methods

Chemicals

All chemicals, including corn starch, soluble starch, maltodextrins,glucose, filter paper were reagent grade or higher and purchased fromSigma-Aldrich (St. Louis, Mo., USA) or Fisher Scientific (Pittsburgh,Pa., USA), unless otherwise noted. Restriction enzymes, T4 ligase, andPhusion DNA polymerase were purchased from New England Biolabs (Ipswich,Mass., USA). Oligonucleotides were synthesized either by Integrated DNATechnologies (Coralville, Iowa, USA) or Eurofins MWG Operon (Huntsville,Ala., USA). Regenerated amorphous cellulose used in enzyme purificationwas prepared from Avicel PH105 (FMC BioPolymer, Philadelphia, Pa., USA)through its dissolution and regeneration, as described in: Ye et al.,Fusion of a family 9 cellulose-binding module improves catalyticpotential of Clostridium thermocellum cellodextrin phosphorylase oninsoluble cellulose. Appl. Microbiol. Biotechnol. 2011; 92:551-560.Escherichia coli Sig10 (Sigma-Aldrich, St. Louis, Mo., USA) was used asa host cell for DNA manipulation and E. coli BL21 (DE3) (Sigma-Aldrich,St. Louis, Mo., USA) was used as a host cell for recombinant proteinexpression. ZYM-5052 media including either 100 mg L⁻¹ ampicillin or 50mg L⁻¹ kanamycin was used for E. coli cell growth and recombinantprotein expression. Cellulase from Trichoderma reesei (Catalog number:C2730) and pullulanase (Catalog number: P1067) were purchased fromSigma-Aldrich (St. Louis, Mo., USA) and produced by Novozymes(Franklinton, N.C., USA). Maltose phosphorylase (Catalog number: M8284)was purchased from Sigma-Aldrich.

Production and Purification of Recombinant Enzymes

The E. coli BL21 (DE3) strain harboring a protein expression plasmid wasincubated in a 1-L Erlenmeyer flask with 100 mL of ZYM-5052 mediacontaining either 100 mg L⁻¹ ampicillin or 50 mg L⁻¹ kanamycin. Cellswere grown at 37° C. with rotary shaking at 220 rpm for 16-24 hours. Thecells were harvested by centrifugation at 12° C. and washed once witheither 20 mM phosphate buffered saline (pH 7.5) containing 50 mM NaCland 5 mM MgCl₂ (heat precipitation and cellulose-binding module) or 20mM phosphate buffered saline (pH 7.5) containing 300 mM NaCl and 5 mMimidazole (Ni purification). The cell pellets were re-suspended in thesame buffer and lysed by ultra-sonication (Fisher Scientific SonicDismembrator Model 500; 5 s pulse on and 10 s off, total 21 min at 50%amplitude). After centrifugation, the target proteins in thesupernatants were purified.

Three approaches were used to purify the various recombinant proteins.His-tagged proteins were purified by the Ni Sepharose 6 Fast Flow resin(GE Life Sciences, Marlborough, Mass., USA). Fusion proteins containinga cellulose-binding module (CBM) and self-cleavage intein were purifiedthrough high-affinity adsorption on a large surface-area regeneratedamorphous cellulose. Heat precipitation at 70-95° C. for 5-30 min wasused to purify hyperthermostable enzymes. The purity of the recombinantproteins was examined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).

Enzymes Used and their Activity Assays

Alpha-glucan phosphorylase (αGP) from Thermotoga maritima (Uniprot IDG4FEH8) was used. Activity was assayed in 50 mM sodium phosphate buffer(pH 7.2) containing 1 mM MgCl₂, and 30 mM maltodextrin at 50° C. Thereaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO) (Vivaproducts, Inc., Littleton, Mass., USA).Glucose 1-phosphate (G1P) was measured using a glucose hexokinase/G6PDHassay kit (Sigma Aldrich, Catalog No. GAHK20-1KT) supplemented with 25U/mL phosphoglucomutase. A unit (U) is described as μmol/min.

Phosphoglucomutase (PGM) from Thermococcus kodakaraensis (Uniprot IDQ68BJ6) was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂ and 5 mM G1P at 50° C. The reaction was stoppedvia filtration of enzyme with a Vivaspin 2 concentrator (10,000 MWCO).The product glucose 6-phosphate (G6P) was determined using ahexokinase/G6PDH assay kit (Sigma Aldrich, Catalog No. GAHK20-1KT).

Two different sources of phosphoglucoisomerase (PGI) were used fromClostridium thermocellum (Uniprot ID A3DBX9) and Thermus thermophilus(Uniprot ID Q5SLL6). Activity was measured in 50 mM HEPES buffer (pH7.2) containing 5 mM MgCl₂ and 10 mM G6P at 50° C. The reaction wasstopped via filtration of enzyme with a Vivaspin 2 concentrator (10,000MWCO). The product, fructose 6-phosphate (F6P), was determined using afructose 6-phosphate kinase (F6PK)/pyruvate dehydrogenase (PK)/lactatedehydrogenase (LD) coupled enzyme assay where a decrease in absorbanceat 340 nm indicates production of F6P. This 200 μM reaction contained 50mM HEPES (pH 7.2), 5 mM MgCl₂, 10 mM G6P, 1.5 mM ATP, 1.5 mM phosphoenolpyruvate, 200 μM NADH, 0.1 U PGI, 5 U PK, and 5 U LD.

The recombinant cellodextrin phosphorylase and cellobiose phosphorylasefrom C. thermocellum are described in Ye et al. Spontaneous high-yieldproduction of hydrogen from cellulosic materials and water catalyzed byenzyme cocktails. ChemSusChem 2009; 2:149-152. Their activities wereassayed as described.

The recombinant polyphosphate glucokinase from Thermobifida fusca YX isdescribed in Liao et al., One-step purification and immobilization ofthermophilic polyphosphate glucokinase from Thermobilida fusca YX:glucose-6-phosphate generation without ATP. Appl. Microbiol. Biotechnol.2012; 93:1109-1117. Its activities were assayed as described.

The recombinant isoamylase from Sulfolobus tokodaii is described inCheng et al., Doubling power output of starch biobattery treated by themost thermostable isoamylase from an archaeon Sulfolobus tokodaii.Scientific Reports 2015; 5:13184. Its activities were assayed asdescribed.

The recombinant 4-alpha-glucanoltransferase from Thermococcus litoralisis described in Jeon et al. 4-α-Glucanotransferase from theHyperthermophilic Archaeon Thermococcus Litoralis. Eur. J. Biochem.1997; 248:171-178. Its activity was measured as described.

Sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum(Uniprot ID D9TT09) was used (Verhaeghe et al. The quest for athermostable sucrose phosphorylase reveals sucrose 6′-phosphatephosphorylase as a novel specificity. Appl Microbiol Biotechnol. 2014August; 98(16):7027-37). Its activity was measured in 50 mM HEPES buffer(pH 7.5) containing 10 mM sucrose and 12 mM organic phosphate. Glucose1-phosphate (G1P) was measured using a glucose hexokinase/G6PDH assaykit supplemented with 25 U/mL phosphoglucomutase as with alpha-glucanphosphorylase.

Psicose 6-phosphate 3-epimerase (P6PE) from Thermoanaerobacteriumthermosaccharolyticum (Uniprot ID D9TQJ4) was used. Activity wasmeasured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl₂, 500 μMCoCl₂, 1 U/mL P6PP, and 10 mM F6P at 50° C. The reaction was stopped viafiltration of enzyme with a Vivaspin 2 concentrator (10,000 MWCO). Theproduct, psicose 6-phosphate (P6P), was determined using Psicose6-phosphate phosphatase and detecting free phosphate release. To detectfree phosphate release, 500 μL of a solution containing 0.1 M zincacetate and 2 mM ammonium molybdate (pH 5) was added to 50 μL ofreaction. This was mixed and followed by 125 μL of 5% ascorbic acid (pH5). This solution was mixed then incubated at 30° C. for 20 min. Theabsorbance at 850 nm was read to determine free phosphate release.Psicose was then verified via HPLC using an Agilent Hi-Plex H-column(sample and control run with 5 mM H₂SO₄ at 0.6 mL/min and 65° C.)

Allose 6-phosphate isomerase (A6PI) from Clostridium thermocellum(Uniprot ID W4V2C8) with the amino acid sequence set forth in SEQ ID NO:1 was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂, 500 μM CoCl₂, 1 U/mL P6PE, 1 U/mL A6PP, and 10 mMF6P at 50° C. The reaction was stopped via filtration of enzyme with aVivaspin 2 concentrator (10,000 MWCO). The product, allose 6-phosphate(P6P), was determined using allose 6-phosphate phosphatase and detectingfree phosphate release as described for P6PE. Allose verified via HPLCthe same as psicose. Another A6P1, such as A6PI from Symbiobacteriumthermophilum (Uniprot ID Q67LX4) with the amino acid sequence set forthin SEQ ID NO: 2, may be used.

Allose 6-phosphate phosphatase (A6PP) from Rubellimicrobium thermophilum(Uniprot ID S9SDA3) with the amino acid sequence set forth in SEQ ID NO:3 was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂, 500 μM CoCl₂, 1 U/mL P6PE, 1 U/mL A6P1, and 10 mMF6P at 50° C. The reaction was stopped via filtration of enzyme with aVivaspin 2 concentrator (10,000 MWCO). The product, allose, wasdetermined by detecting free phosphate release as described for P6PE.Allose verified via HPLC the same as psicose. Other A6PPs, such as A6PPfrom Thermotoga maritima (Uniprot ID Q9X0Y1) with the amino acidsequence set forth in SEQ ID NO: 4, A6PP from Thermoanaerobacteriumsaccharolyticum (Uniprot ID 13VT81) with the amino acid sequence setforth in SEQ ID NO: 5, A6PP from Streptomyces thermoautotrophicus(Uniprot ID A0A132NF06) with the amino acid sequence set forth in SEQ IDNO: 6, and A6PP from Sphaerobacter thermophilus (Uniprot ID D1C7G9) withthe amino acid sequence set forth in SEQ ID NO: 7, may be used.

Mannose 6-phosphate isomerase (M6PI) from Pseudonocardia thermophila(Uniprot ID A0A1M6TLY7) with the amino acid sequence set forth in SEQ IDNO: 8 was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂, 1 U/mL PGI, 1 U/mL M6PP, and 10 mM F6P at 50° C.The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, mannose 6-phosphate (M6P), wasdetermined using mannose 6-phosphate phosphatase (M6PP) and detectingfree phosphate release as described for P6PE. Mannose verified via HPLCthe same as psicose. Other M6PIs such as M6PI from Caldithrix abyssi(Uniprot ID H1XQS6) with the amino acid sequence set forth in SEQ ID NO:9, M6PI from Myceliophthora thermophila (Uniprot ID G2Q982) with theamino acid sequence set forth in SEQ ID NO: 10 and M6PI from Treponemacaldarium (Uniprot ID F8F1Z8) with the amino acid sequence set forth inSEQ ID NO: 11 may be used.

Mannose 6-phosphate phosphatase (M6PP) from Tepidimonas fonticaldi(Uniprot ID A0A1A6DS13) with the amino acid sequence set forth in SEQ IDNO: 12 was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂, and 10 mM mannose 6-phosphate at 50° C. Thereaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, mannose, was determined bydetecting free phosphate release as described for P6PE. Mannose verifiedvia HPLC the same as psicose. Other M6PP such as M6PP from Thermomonashydrothermalis (Uniprot ID A0A1M4UN08) with the amino acid sequence setforth in SEQ ID NO: 13 and M6PP from Sulfurivirga caldicuralii (UniprotID A0AlN6FCW3) with the amino acid sequence set forth in SEQ ID NO: 14may be used.

Bifunctional phosphoglucose/phosphomannose isomerase (PGPMI) fromSyntrophothermus lipocalidus (Uniprot ID D7CPH7) with the amino acidsequence set forth in SEQ ID NO: 15 was used. Activity was measured in50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl₂, 1 U/mL M6PP, and 10mM G6P at 50° C. The reaction was stopped via filtration of enzyme witha Vivaspin 2 concentrator (10,000 MWCO). The product, M6P, wasdetermined using M6PP and detecting free phosphate release as describedfor P6PE. Mannose verified via HPLC the same as psicose. Other PGPMIsuch as PGPMI from Schleiferia thermophila (Uniprot ID A0A085L170) withthe amino acid sequence set forth in SEQ ID NO: 16 and PGPMI fromThermodesulfobium narugense (Uniprot ID M1E6Z3) with the amino acidsequence set forth in SEQ ID NO: 17 may be used.

Galactose 6-phosphate isomerase (Gal6PI) from Lactococcus lactis(obligate dimer; Uniprot IDs P23494 and P23495 with the amino acidsequences set forth in SEQ ID NO: 18 and 19, respectively) is used (vanRooijen et al. Molecular Cloning, Characterization, and NucleotideSequence of the Tagatose 6-Phosphate Pathway Gene Cluster of the LactoseOperon of Lactococcus Zactis. J. Biol. Chem. 1991; 266:7176-7181).Activity is measured in 50 mM HEPES buffer (pH 7.2) containing 5 mMMgCl₂, 1 U/mL fructose 6-phosphate 4-epimerase (F6PE), 1 U/mL galactose6-phosphate phosphatase (Gal6PP), and 10 mM fructose 6-phosphate at 37°C. The reaction is stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, galactose 6-phosphate (gal6P),is determined using Gal6PP and detecting free phosphate release asdescribed for P6PE. Galactose verified via HPLC the same as psicose.

Galactose 6-phosphate phosphatase (Gal6PP) from Bacteroidesthetaiotaomicron (Uniprot ID Q8A2F3) with the amino acid sequence setforth in SEQ ID NO: 20 was used. Activity was measured in 50 mM HEPESbuffer (pH 7.2) containing 5 mM MgCl₂, and 10 mM galactose 6-phosphateat 50° C. The reaction was stopped via filtration of enzyme with aVivaspin 2 concentrator (10,000 MWCO). The product, galactose, wasdetermined by detecting free phosphate release as described for P6PE.Galactose verified via HPLC the same as psicose.

Fructose 6-phosphate phosphatase (F6PP) from Halothermothrix orenii(Uniprot ID B8CWV3) with the amino acid sequence set forth in SEQ ID NO:21 was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂, and 10 mM fructose 6-phosphate at 50° C. Thereaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, fructose, was determined bydetecting free phosphate release as described for P6PE. Fructoseverified via HPLC the same as psicose.

Tagatose 6-phosphate phosphatase (TEPP) from Archaeoglobus fugidis(Uniprot ID A0A075WB87) was used. Activity was measured in 50 mM HEPESbuffer (pH 7.2) containing 5 mM MgCl₂ and 10 mM T6P at 50° C. Thereaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). Tagatose production was determined bydetecting free phosphate release as described for F6PE.

Psicose 6-phosphate phosphatase (P6PP) from Clostridium thermocellum(UNIPROT ID A3DC21), was used. Activity was measured in 50 mM HEPESbuffer (pH 7.2) containing 5 mM MgCl2, 80 μM CoCl2, 1 U/mL P6PE, and 10mM F6P at 50° C. The reaction was stopped via filtration of enzyme witha Vivaspin 2 concentrator (10,000 MWCO). The product, psicose, wasdetermined through detecting free phosphate release as described forP6PE.

Enzyme units used in each Example below can be increased or decreased toadjust the reaction time as desired. For example, if one wanted toperform Example 9 in 8 h instead of 24 h, the units of the enzymes wouldbe increased about 3-fold. Conversely, if one wanted perform example 9in 48 h instead of 24 h the enzyme units could be decreased about2-fold. These examples illustrate how the amount of enzyme units can beused to increase or decrease reaction time while maintaining constantproductivity.

All Products

Example 1

To validate the technical feasibility of the enzymatic biosynthesis offructose 6-phosphate from starch, three enzymes were recombinantlyexpressed: alpha-glucan phosphorylase from T. maritima (Uniprot IDG4FEH8), phosphoglucomutase from Thermococcus kodakaraensis (Uniprot IDQ68BJ6), and phosphoglucoisomerase from Clostridium thermocellum(Uniprot ID A3DBX9). The recombinant proteins were over-expressed in E.coli BL21 (DE3) and purified as described above.

A 0.20 mL reaction mixture containing 10 g/L soluble starch, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 0.5 mM ZnCl₂, 0.01 U ofαGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours.The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), wasdetermined using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P asdescribed above. The final concentration of F6P after 24 hours was 3.6g/L.

Example 2

Same tests as in Example 1 (other than reaction temperatures) werecarried out from 40 to 80° C. It was found that 10 g/L soluble starchproduced 0.9 g/L F6P at 40° C. and 3.6 g/L F6P at 80° C. after 40 hourreactions. These results suggest that increasing reaction temperaturefor this set of enzymes increased F6P yields, but too high oftemperature may impair some enzyme activity.

Example 3

It was found that, at 80° C., an enzyme ratio of αGP: PGM: PGI ofapproximately 1:1:1 resulted in fast F6P generation. It was noted thatthe enzyme ratio did not influence final F6P concentration greatly ifthe reaction time was long enough. However, the enzyme ratio affectsreaction rates and the total cost of enzymes used in the system.

Example 4

A 0.20 mL reaction mixture containing 10 g/L maltodextrin, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 0.5 mM ZnCl₂, 0.01 U ofαGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours.The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), wasdetermined using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P asdescribed above. The final concentration of F6P after 24 hours was 3.6g/L.

Example 5

To test for F6P production from Avicel, Sigma cellulase was used tohydrolyze cellulose at 50° C. To remove beta-glucosidase from commercialcellulase, 10 filter paper units/mL of cellulase was mixed to 10 g/LAvicel at an ice-water bath for 10 min. After centrifugation at 4° C.,the supernatant containing beta-glucosidase was decanted. Avicel thatwas bound with cellulase containing endoglucanase and cellobiohydrolasewas resuspended in a citrate buffer (pH 4.8) for hydrolysis at 50° C.for three days. The cellulose hydrolysate was mixed with 5 U/mLcellodextrin phosphorylase, 5 U/L cellobiose phosphorylase, 5 U/mL ofαGP, 5 U/mL PGM, and 5 U/mL PGI in a 100 mM HEPES buffer (pH 7.2)containing 10 mM phosphate, 5 mM MgCl₂ and 0.5 mM ZnCl₂. The reactionwas conducted at 60° C. for 72 hours and high concentrations of F6P werefound (small amounts of glucose and no cellobiose). F6P was detectedusing the coupled enzyme assay described above. Glucose was detectedusing a hexokinase/G6PDH assay kit as described above.

Example 6

To increase F6P yields from Avicel, Avicel was pretreated withconcentrated phosphoric acid to produce amorphous cellulose (RAC), asdescribed in Zhang et al. A transition from cellulose swelling tocellulose dissolution by o-phosphoric acid: evidence from enzymatichydrolysis and supramolecular structure. Biomacromolecules 2006;7:644-648. To remove beta-glucosidase from commercial cellulase, 10filter paper units/mL of cellulase was mixed with 10 g/L RAC in anice-water bath for 5 min. After centrifugation at 4° C., the supernatantcontaining beta-glucosidase was decanted. The RAC that was bound withcellulase containing endoglucanase and cellobiohydrolase was resuspendedin a citrate buffer (pH 4.8) for hydrolysis at 50° C. for 12 hours. TheRAC hydrolysate was mixed with 5 U/mL cellodextrin phosphorylase, 5 U/mLcellobiose phosphorylase, 5 U/mL of αGP, 5 U/mL PGM, and 5 U/mL PGI in a100 mM HEPES buffer (pH 7.2) containing 10 mM phosphate, 5 mM MgCl₂ and0.5 mM ZnCl₂. The reaction was conducted at 60° C. for 72 hours. Highconcentrations of F6P and glucose were recovered because no enzymes wereadded to convert glucose to F6P. F6P was detected using the coupledenzyme assay described above. Glucose was detected using ahexokinase/G6PDH assay kit as described above.

Example 7

To further increase F6P yields from RAC, polyphosphate glucokinase andpolyphosphate were added. To remove beta-glucosidase from commercialcellulase, 10 filter paper units/mL of cellulase was mixed with 10 g/LRAC in an ice-water bath for 5 min. After centrifugation at 4° C., thesupernatant containing beta-glucosidase was decanted. The RAC that wasbound with cellulase containing endoglucanase and cellobiohydrolase wasre-suspended in a citrate buffer (pH 4.8) for hydrolysis at 50° C. wasincubated in a citrate buffer (pH 4.8) for hydrolysis at 50° C. for 12hours. The RAC hydrolysate was mixed with 5 U/mL polyphosphateglucokinase, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiosephosphorylase, 5 U/mL of αGP, 5 U/mL PGM, and 5 U/mL PGI in a 100 mMHEPES buffer (pH 7.2) containing 50 mM polyphosphate, 10 mM phosphate, 5mM MgCl₂ and 0.5 mM ZnCl₂. The reaction was conducted at 50° C. for 72hours. F6P was found in high concentrations with only small amounts ofglucose now present. F6P was detected using the coupled enzyme assaydescribed above. Glucose was detected using a hexokinase/G6PDH assay kitas described above.

Example 8

To determine the concentration range of phosphate buffered saline (PBS),a 0.20 mL reaction mixture containing 50 g/L maltodextrin; 6.25 mM, 12.5mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline pH 7.2; 5 mMMgCl2; 0.1 U of αGP; 0.1 U PGM; and 0.1 U PGI was incubated at 50° C.for 6 hours. The short duration ensures completion was not reached, andtherefore differences in efficiency can be clearly seen. Production ofF6P was quantified using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P.Respectively, a yield of 4.5 g/L, 5.1 g/L, 5.6 g/L, 4.8 g/L, or 4.9 g/LF6P was obtained for the reactions containing either 6.25 mM, 12.5 mM,25 mM, 37.5 mM, or 50 mM phosphate buffered saline pH 7.2 (Table 1).These results indicate that a concentration of 25 mM PBS pH 7.2 wasideal for these particular reaction conditions. It is important to notethat even the use of 6.25 mM PBS at pH 7.2 results in significantturnover due to phosphate recycling. This shows that the disclosedphosphate recycling methods are able to keep phosphate levels low evenat industrial levels of volumetric productivity (e.g., 200-300 g/Lmaltodextrin).

TABLE 1 Concentration of PBS pH 7.2 (mM) g/L of F6P 6.25 4.5 12.5 5.1 255.6 37.5 4.8 50 4.9

Example 9

To determine the pH range of the cascade reaction, a 0.20 mL reactionmixture containing 50 g/L maltodextrin; 50 mM phosphate buffered salinepH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 7.2, or 7.3; 5 mM MgCl2; 0.02 U of αGP;0.02 U PGM; and 0.02 U PGI was incubated at 50° C. for 16 hours. Theunits are lowered to ensure completion was not reached, and thereforedifferences in efficiency can be clearly seen. Production of F6P wasquantified as in example 12. Respectively, a yield of 4.0 g/L, 4.1 g/L4.2 g/L, 4.1 g/L, 4.4 g/L, 4.1 g/L, 3.8 g/L or 4.0 g/L F6P was obtainedfor reactions containing 50 mM phosphate buffered saline at pH 6.0, 6.2,6.4, 6.6, 6.8, 7.0, 7.2, or 7.3 (Table 2). These results indicate that apH of 6.8 was ideal for these particular reaction conditions, althoughthe system works through a wide pH range.

TABLE 2 pH of PBS g/L of F6P 6.0 4.0 6.2 4.1 6.4 4.2 6.6 4.1 6.8 4.4 7.04.1 7.2 3.8 7.3 4.0

Allose

Example 10

To validate allose production from F6P, 10 g/L F6P was mixed with 1 U/mLP6PE, 1 U/mL A6PI and 1 U/mL A6PP in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂ and 500 μM CoCl₂. The reaction was incubated for 3hours at 50° C. Conversion of F6P to allose was seen via HPLC (Agilent1100 series) using an Agilent Hi-Plex H-column and refractive indexdetector. The sample and control were run in 5 mM H₂SO₄ at 0.6 mL/minand 65° C.

Example 11

To validate production of allose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 500 μM CoCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI,0.05 U P6PE, 0.05 U A6PI and 0.05 U A6PP was incubated at 50° C. for 24hours. The reaction was stopped via filtration of enzyme with a Vivaspin2 concentrator (10,000 MWCO). Allose was verified via HPLC as describedin Example 10.

Example 12

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase was incubated at 80° C. for24 hours. This was used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 500 μM CoCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI,0.05 U P6PE, 0.05 U A6P1, and 0.05 U A6PP was incubated at 50° C. for 24hours. Production of allose was verified as in Example 10.

Example 13

To further increase allose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) was added to the reaction described in Example 11.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 12), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 500 μM CoCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 UP6PE, 0.05 U A6PI, 0.05 U A6PP, and 0.05 U 4GT was incubated at 50° C.for 24 hours. Production of allose was verified as in Example 10.

Example 14

To further increase allose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 11.

Example 15

To further increase allose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 11.

Example 16

To produce allose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 500μM CoCl₂, 0.05 U fructose polyphosphate kinase, 0.05 U P6PE, 0.05 A6P1,and 0.05 U A6PP is incubated at 50° C. for 24 hours. Production ofallose is quantified as in Example 10.

Example 17

To produce allose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 500μM CoCl₂, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U P6PE,0.05 A6P1, and 0.05 U A6PP is incubated at 50° C. for 24 hours.Production of allose is quantified as in Example 10.

Example 18

To produce allose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 500 μMCoCl₂, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U P6PE,0.05 A6P1, and 0.05 U A6PP is incubated at 50° C. for 24 hours.Production of allose is quantified as in Example 10.

Example 19

To further increase yields of allose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inExample 18. Production of allose is quantified as in Example 10.

Mannose

Example 20

To validate mannose production from F6P, 10 g/L F6P was mixed with 1U/mL M6P1/PGPMI, and 1 U/mL M6PP in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂. The reaction was incubated for 3 hours at 50° C.Conversion of F6P to mannose was seen via HPLC (Agilent 1100 series)using an Agilent Hi-Plex H-column and refractive index detector. Thesample and control were run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 21

To validate production of mannose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 UM6P1/PGPMI (no PGI needed in PGPMI case), and 0.05 U M6PP was incubatedat 50° C. for 24 hours. The reaction was stopped via filtration ofenzyme with a Vivaspin 2 concentrator (10,000 MWCO). Mannose wasverified via HPLC as described in Example 20.

Example 22

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase was incubated at 80° C. for24 hours. This was used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 UM6P1/PGPMI (no PGI needed in PGPMI case), and 0.05 U M6PP was incubatedat 50° C. for 24 hours. Production of mannose was verified as in Example20.

Example 23

To further increase mannose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) was added to the reaction described in Example 21.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 22), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U M6P1/PGPMI (noPGI needed in PGPMI case), 0.05 U M6PP, and 0.05 U 4GT was incubated at50° C. for 24 hours. Production of mannose was verified as in Example20.

Example 24

To further increase mannose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 21.

Example 25

To further increase mannose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 21.

Example 26

To produce mannose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂,0.05 U fructose polyphosphate kinase, 0.05 U M6P1/PGPMI (no PGI neededin PGPMI case), and 0.05 U M6PP is incubated at 50° C. for 24 hours.Production of mannose is quantified as in Example 20.

Example 27

To produce mannose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U M6PI/PGPMI (no PGIneeded in PGPMI case), and 0.05 U M6PP is incubated at 50° C. for 24hours. Production of mannose is quantified as in Example 20.

Example 28

To produce mannose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U M6P1/PGPMI (no PGIneeded in PGPMI case), and 0.05 U M6PP is incubated at 50° C. for 24hours. Production of mannose is quantified as in Example 20.

Example 29

To further increase yields of mannose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inExample 28. Production of mannose is quantified as in Example 20.

Galactose

Example 30

To validate galactose production from F6P, 10 g/L F6P is mixed with 1U/mL Gal6PI, and 1 U/mL Gal6PP in 50 mM HEPES buffer (pH 7.2) containing5 mM MgCl₂. The reaction is incubated for 3 hours at 37° C. Conversionof F6P to galactose is seen via HPLC (Agilent 1100 series) using anAgilent Hi-Plex H-column and refractive index detector. The sample andcontrol are run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 31

To validate production of galactose from maltodextrin, a 0.20 mLreaction mixture containing 20 g/L maltodextrin, 50 mM phosphatebuffered saline pH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 UPGI, 0.05 U Gal6PI, and 0.05 U Gal6PP is incubated at 37° C. for 24hours. The reaction is stopped via filtration of enzyme with a Vivaspin2 concentrator (10,000 MWCO). Galactose is verified via HPLC asdescribed in Example 30.

Example 32

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase is incubated at 80° C. for24 hours. This is used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI,and 0.05 U Gal6PP is incubated at 37° C. for 24 hours. Production ofgalactose is verified as in Example 30.

Example 33

To further increase galactose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in Example 31.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 12), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI, 0.05 UGal6PP, and 0.05 U 4GT is incubated at 37° C. for 24 hours. Productionof galactose is verified as in Example 30.

Example 34

To further increase galactose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 31.

Example 35

To further increase galactose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 31.

Example 36

To produce galactose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂,0.05 U fructose polyphosphate kinase, 0.05 U Gal6PI, and 0.05 U Gal6PPis incubated at 37° C. for 24 hours. Production of galactose isquantified as in Example 30.

Example 37

To produce galactose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U Gal6PI, and 0.05 UGal6PP is incubated at 37° C. for 24 hours. Production of galactose isquantified as in Example 30.

Example 38

To produce galactose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U Gal6PI, and 0.05 UGal6PP is incubated at 37° C. for 24 hours. Production of galactose isquantified as in Example 30.

Example 39

To further increase yields of galactose from sucrose, 75 mMpolyphosphate and 0.05 polyphosphate fructokinase is added to thereaction mixture in Example 38. Production of galactose is quantified asin Example 30.

Example 40

To validate galactose production from Gal6P, 10 g/L Gal6P was mixed with1 U/mL Gal6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl₂. Thereaction was incubated for 1 hour at 50° C. Conversion of Gal6P togalactose is seen free phosphate detection. To detect free phosphaterelease, 500 μL of a solution containing 0.1 M zinc acetate and 2 mMammonium molybdate (pH 5) was added to 50 μL of reaction. This was mixedand followed by 125 μL of 5% ascorbic acid (pH 5). This solution wasmixed then incubated at 30° C. for 20 min. The absorbance at 850 nm wasread to determine free phosphate release.

Fructose

Example 41

To validate fructose production from F6P, 10 g/L F6P was mixed with 1U/mL F6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl₂. Thereaction was incubated for 3 hours at 50° C. Conversion of F6P tofructose was seen via HPLC (Agilent 1100 series) using an AgilentHi-Plex H-column and refractive index detector. The sample and controlwere run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 42

To validate production of fructose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, and 0.05 UF6PP was incubated at 50° C. for 24 hours. The reaction was stopped viafiltration of enzyme with a Vivaspin 2 concentrator (10,000 MWCO).Fructose was verified via HPLC as described in Example 41.

Example 43

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase was incubated at 80° C. for24 hours. This was used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, and 0.05 U F6PPwas incubated at 50° C. for 24 hours. Production of fructose wasverified as in Example 41.

Example 44

To further increase fructose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) was added to the reaction described in Example 42.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 12), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PP, and 0.05 U4GT was incubated at 50° C. for 24 hours. Production of fructose wasverified as in Example 41.

Example 45

To further increase fructose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 42.

Example 46

To further increase fructose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 42.

Example 47

To produce fructose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, and 0.05 U F6PP is incubatedat 50° C. for 24 hours. Production of fructose is quantified as inExample 41.

Example 48

To produce fructose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, and 0.05 U F6PP wasincubated at 50° C. for 24 hours. Production of fructose was quantifiedas in Example 41.

Altrose

Example 49

To validate altrose production from F6P, 10 g/L F6P is mixed with 1 U/mLP6PE, 1 U/mL altrose 6-phosphate isomerase (Alt6PI), and 1 U/mL altrose6-phosphate phosphatase (Alt6PP) in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂. The reaction is incubated for 3 hours at 50° C.Conversion of F6P to altrose is seen via HPLC (Agilent 1100 series)using an Agilent Hi-Plex H-column and refractive index detector. Thesample and control are run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 50

To validate production of altrose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE,0.05 U Alt6PI, and 0.05 U Alt6PP is incubated at 50° C. for 24 hours.The reaction is stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). Altrose is verified via HPLC as described inExample 49.

Example 51

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase is incubated at 80° C. for24 hours. This is used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE,0.05 U Alt6PI, and 0.05 U Alt6PP is incubated at 50° C. for 24 hours.Production of altrose is verified as in Example 49.

Example 52

To further increase altrose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in Example 50.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 50), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 UAlt6PI, 0.05 U Alt6PP, and 0.05 U 4GT is incubated at 50° C. for 24hours. Production of altrose is verified as in Example 49.

Example 53

To further increase altrose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 50.

Example 54

To further increase altrose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 50.

Example 55

To produce altrose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂,0.05 U fructose polyphosphate kinase, 0.05 U P6PE, 0.05 U Alt6PI, and0.05 U Alt6PP is incubated at 50° C. for 24 hours. Production of altroseis quantified as in Example 49.

Example 56

To produce altrose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI,and 0.05 U Alt6PP is incubated at 50° C. for 24 hours. Production ofaltrose is quantified as in Example 49.

Example 57

To produce altrose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI,and 0.05 U Alt6PP is incubated at 50° C. for 24 hours. Production ofaltrose is quantified as in Example 49.

Example 58

To further increase yields of altrose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inExample 56. Production of altrose is quantified as in Example 49.

Talose

Example 59

To validate talose production from F6P, 10 g/L F6P is mixed with 1 U/mLF6PE, 1 U/mL talose 6-phosphate isomerase (Tal6PI), and 1 U/mL talose6-phosphate phosphatase (Tal6PP) in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂. The reaction is incubated for 3 hours at 50° C.Conversion of F6P to talose is seen via HPLC (Agilent 1100 series) usingan Agilent Hi-Plex H-column and refractive index detector. The sampleand control are run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 60

To validate production of talose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,0.05 U Tal6PI, and 0.05 U Tal6PP is incubated at 50° C. for 24 hours.The reaction is stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). Talose is verified via HPLC as described inExample 59.

Example 61

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase is incubated at 80° C. for24 hours. This is used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,0.05 U Tal6PI, and 0.05 U Tal6PP is incubated at 50° C. for 24 hours.Production of talose is verified as in Example 59.

Example 62

To further increase talose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in Example 60.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 60), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 UTal6PI, 0.05 U Tal6PP, and 0.05 U 4GT is incubated at 50° C. for 24hours. Production of talose is verified as in Example 59.

Example 63

To further increase talose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 59.

Example 64

To further increase talose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 60.

Example 65

To produce talose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂,0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U Tal6PI, and0.05 U Tal6PP is incubated at 50° C. for 24 hours. Production of taloseis quantified as in Example 59.

Example 66

To produce talose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI,and 0.05 U Tal6PP is incubated at 50° C. for 24 hours. Production oftalose is quantified as in Example 59.

Example 67

To produce talose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI,and 0.05 U Tal6PP is incubated at 50° C. for 24 hours. Production oftalose is quantified as in Example 59.

Example 68

To further increase yields of talose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inExample 66. Production of talose is quantified as in Example 59.

Sorbose

Example 69

To validate sorbose production from F6P, 10 g/L F6P is mixed with 1 U/mLF6PE, 1 U/mL sorbose 6-phosphate 3-epimerase (S6PE), and 1 U/mL sorbose6-phosphate phosphatase (S6PP) in 50 mM HEPES buffer (pH 7.2) containing5 mM MgCl₂. The reaction is incubated for 3 hours at 50° C. Conversionof F6P to sorbose is seen via HPLC (Agilent 1100 series) using anAgilent Hi-Plex H-column and refractive index detector. The sample andcontrol are run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 70

To validate production of sorbose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,0.05 U S6PE, and 0.05 U S6PP is incubated at 50° C. for 24 hours. Thereaction is stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). Sorbose is verified via HPLC as described inExample 68.

Example 71

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase is incubated at 80° C. for24 hours. This is used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,0.05 U S6PE, and 0.05 U S6PP is incubated at 50° C. for 24 hours.Production of sorbose is verified as in Example 69.

Example 72

To further increase sorbose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in Example 70.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 70), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 US6PE, 0.05 U S6PP, and 0.05 U 4GT is incubated at 50° C. for 24 hours.Production of sorbose is verified as in Example 69.

Example 73

To further increase sorbose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 70.

Example 74

To further increase sorbose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 69.

Example 75

To produce sorbose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂,0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, and 0.05U S6PP is incubated at 50° C. for 24 hours. Production of sorbose isquantified as in Example 69.

Example 76

To produce sorbose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE,and 0.05 U S6PP is incubated at 50° C. for 24 hours. Production ofsorbose is quantified as in Example 69.

Example 77

To produce sorbose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE,and 0.05 U S6PP is incubated at 50° C. for 24 hours. Production ofsorbose is quantified as in Example 69.

Example 78

To further increase yields of sorbose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inExample 76. Production of sorbose is quantified as in Example 69.

Gulose

Example 79

To validate gulose production from F6P, 10 g/L F6P is mixed with 1 U/mLF6PE, 1 U/mL S6PE, 1 U/mL gulose 6-phosphate isomerase (Gul6PI), and 1U/mL gulose 6-phosphate phosphatase (Gul6PP) in 50 mM HEPES buffer (pH7.2) containing 5 mM MgCl₂. The reaction is incubated for 3 hours at 50°C. Conversion of F6P to gulose is seen via HPLC (Agilent 1100 series)using an Agilent Hi-Plex H-column and refractive index detector. Thesample and control are run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 80

To validate production of gulose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP is incubated at 50° C. for24 hours. The reaction is stopped via filtration of enzyme with aVivaspin 2 concentrator (10,000 MWCO). Gulose is verified via HPLC asdescribed in Example 79.

Example 81

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase is incubated at 80° C. for24 hours. This is used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP is incubated at 50° C. for24 hours. Production of gulose is verified as in Example 79.

Example 82

To further increase gulose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in Example 80.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 80), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI0.05 U F6PE, 0.05 U S6PE,0.05 U Gul6PI, 0.05 U Gul6PP, and 0.05 U 4GT is incubated at 50° C. for24 hours. Production of gulose is verified as in Example 79.

Example 83

To further increase gulose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 80.

Example 84

To further increase gulose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 80.

Example 85

To produce gulose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂,0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, 0.05 UGul6PI, and 0.05 U Gul6PP is incubated at 50° C. for 24 hours.Production of gulose is quantified as in Example 79.

Example 86

To produce gulose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE,0.05 U Gul6PI, and 0.05 U Gul6PP is incubated at 50° C. for 24 hours.Production of gulose is quantified as in Example 79.

Example 87

To produce gulose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE,0.05 U Gul6PI, and 0.05 U Gul6PP is incubated at 50° C. for 24 hours.Production of gulose is quantified as in Example 79.

Example 88

To further increase yields of gulose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inExample 86. Production of gulose is quantified as in Example 79.

Idose

Example 89

To validate idose production from F6P, 10 g/L F6P is mixed with 1 U/mLF6PE, 1 U/mL S6PE, 1 U/mL idose 6-phosphate isomerase (16PI), and 1 U/mLidose 6-phosphate phosphatase (16PP) in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂. The reaction is incubated for 3 hours at 50° C.Conversion of F6P to idose is seen via HPLC (Agilent 1100 series) usingan Agilent Hi-Plex H-column and refractive index detector. The sampleand control are run in 5 mM H₂SO₄ at 0.6 mL/min and 65° C.

Example 90

To validate production of idose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,0.05 U S6PE, 0.05 U 16P1, and 0.05 U 16PP is incubated at 50° C. for 24hours. The reaction is stopped via filtration of enzyme with a Vivaspin2 concentrator (10,000 MWCO). Idose is verified via HPLC as described inExample 89.

Example 91

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, and 0.1 g/L isoamylase is incubated at 80° C. for24 hours. This is used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PG1, 0.05 U F6PE,0.05 U S6PE, 0.05 U I6P1, and 0.05 U 16PP is incubated at 50° C. for 24hours. Production of idose is verified as in Example 89.

Example 92

To further increase idose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in Example 90.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see Example 90), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI0.05 U F6PE, 0.05 U S6PE,0.05 U 16PI, 0.05 U 16PP, and 0.05 U 4GT is incubated at 50° C. for 24hours. Production of idose is verified as in Example 89.

Example 93

To further increase idose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 90.

Example 94

To further increase idose yields from maltodextrin, 0.05 U polyphosphateglucokinase and 75 mM polyphosphate is added to the reaction describedin Example 90.

Example 95

To produce idose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂,0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, 0.05 U16P1, and 0.05 U 16PP is incubated at 50° C. for 24 hours. Production ofidose is quantified as in Example 89.

Example 96

To produce idose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE,0.05 U 16P1, and 0.05 U 16PP is incubated at 50° C. for 24 hours.Production of idose is quantified as in Example 89.

Example 97

To produce idose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE,0.05 U 16P1, and 0.05 U 16PP is incubated at 50° C. for 24 hours.Production of idose is quantified as in Example 89.

Example 98

To further increase yields of idose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inExample 96. Production of idose is quantified as in Example 89.

Tagatose

Example 99

To validate tagatose production from F6P, 2 g/L F6P was mixed with 1U/ml fructose 6-phosphate epimerase (F6PE) and 1 U/ml tagatose6-phosphate phosphatase (T6PP) in 50 mM HEPES buffer (pH 7.2) containing5 mM MgCl2. The reaction was incubated for 16 hours at 50° C. 100%conversion of F6P to tagatose is seen via HPLC (Agilent 1100 series)using an Agilent Hi-Plex H-column and refractive index detector. Thesample was run in 5 mM H2SO4 at 0.6 mL/min.

Example 100

To validate production of tagatose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl2, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE,and 0.05 U T6PP was incubated at 50° C. for 24 hours. The reaction wasstopped via filtration of enzyme with a Vivaspin 2 concentrator (10,000MWCO). Tagatose was detected and quantified using an Agilent 1100 seriesHPLC with refractive index detector and an Agilent Hi-Plex H-column. Themobile phase was 5 mM H2SO4, which ran at 0.6 mL/min. A yield of 9.2 g/Ltagatose was obtained. This equates to 92% of the theoretical yield dueto limits of maltodextrin degradation without enzymes such as isoamylaseor 4-glucan transferase. Standards of various concentrations of tagatosewere used to quantify our yield.

Example 101

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl2, and 0.1 g/L isoamylase was incubated at 80° C. for24 hours. This was used to create another reaction mixture containing 20g/L isoamylase treated maltodextrin, 50 mM phosphate buffered saline pH7.2, 5 mM MgCl2, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, and0.05 U T6PP was incubated at 50° C. for 24 hours. Production of tagatosewas quantified as in Example 99. The yield of tagatose was increased to16 g/L with the pretreatment of maltodextrin by isoamylase. This equatesto 80% of the theoretical yield.

Example 102

To further increase tagatose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) was added to the reaction described in Example 100.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see example 9), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl2, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 UT6PP, and 0.05 U 4GT was incubated at 50° C. for 24 hours. Production oftagatose was quantified as in example 9. The yield of tagatose wasincreased to 17.7 g/L with the addition of 4GT to IA-treatedmaltodextrin. This equates to 88.5% of the theoretical yield.

Example 103

To investigate scale-up, a 20 mL reaction mixture containing 50 g/Lisoamylase treated maltodextrin (see Example 99), 50 mM phosphatebuffered saline pH 7.2, 5 mM MgCl2, 10 U of αGP, 10 U PGM, 10 U PGI, 10U F6PE, and 10 U T6PP was incubated at 50° C. for 24 hours. Productionof tagatose was quantified as in example 8. The yield of tagatose was37.6 g/L at the 20 mL scale and 50 g/L maltodextrin. This equates to 75%of the theoretical yield. These results indicate that scale-up to largerreaction volumes will not result in significant loses of yield.

Example 104

To further increase tagatose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 100.

Example 105

To further increase tagatose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 99.

Example 106

To produce tagatose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2,0.05 U fructose polyphosphate kinase, 0.05 U F6PE, and 0.05 U T6PP isincubated at 50° C. for 24 hours. Production of tagatose is quantifiedas in Example 100.

Example 107

To produce tagatose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, and 0.05 U T6PPis incubated at 50° C. for 24 hours. Production of tagatose isquantified as in Example 100.

Example 108

To produce tagatose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 0.05 Usucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, and 0.05 UT6PP is incubated at 50° C. for 24 hours. Production of tagatose isquantified as in Example 100.

Example 109

To further increase yields of tagatose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inexample 15. Production of tagatose is quantified as in Example 100.

Psicose

Example 110

To validate psicose production from F6P, 2 g/L F6P was mixed with 1 U/mlP6PE and 1 U/ml P6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mMMgCl₂ and 80 μM CoCl₂. The reaction was incubated for 6 hours at 50° C.99% conversion of F6P to psicose was seen via HPLC (Agilent 1100 series)using an Agilent Hi-Plex H-column and refractive index detector. Thesample was run in 5 mM H₂SO₄ at 0.6 mL/min.

Example 111

To validate production of psicose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 80 μM CoCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI,0.05 U P6PE and 0.05 U P6PP is incubated at 50° C. for 24 hours. Thereaction is stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). Psicose is detected and quantified using anAgilent 1100 series HPLC with refractive index detector and an AgilentHi-Plex H-column. The mobile phase is 5 mM H₂SO₄, which runs at 0.6mL/min. Standards of various concentrations of psicose are used toquantify our yield.

Example 112

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, 80 μM CoCl₂, and 0.1 g/L isoamylase is incubatedat 80° C. for 24 hours. This is used to create another reaction mixturecontaining 20 g/L isoamylase treated maltodextrin, 50 mM phosphatebuffered saline pH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 UPGI, 0.05 U P6PE, and 0.05 U P6PP is incubated at 50° C. for 24 hours.Production of psicose is quantified as in Example 111.

Example 113

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 4.5), 5 mM MgCl₂, and 1:200 dilution of Novozymes D6 pullulanase isincubated at 50° C. for 4 hours. This is used to create another reactionmixture containing 20 g/L pullulanase treated maltodextrin, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 80 μM CoCl₂, 0.05 U ofαGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP is incubatedat 50° C. for 24 hours. Production of psicose is quantified as inExample 111.

Example 114

To further increase psicose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in Example 111.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see example 9), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 80 μM CoCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 UP6PE, 0.05 U P6PP, and 0.05 U 4GT is incubated at 50° C. for 24 hours.Production of psicose is quantified as in Example 111.

Example 115

To investigate scale-up, a 20 mL reaction mixture containing 50 g/Lisoamylase treated maltodextrin (see Example 10), 50 mM phosphatebuffered saline pH 7.2, 5 mM MgCl₂, 80 μM CoCl₂, 10 U of αGP, 10 U PGM,10 U PGI, 10 U P6PE, and 10 U P6PP is incubated at 50° C. for 24 hours.Production of psicose was quantified as in Example 111.

Example 116

To further increase psicose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 110.

Example 117

To further increase psicose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 111.

Example 118

To produce psicose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 80μM CoCl2, 0.05 U fructose polyphosphate kinase, 0.05 U P6PE, and 0.05 UP6PP is incubated at 50° C. for 24 hours. Production of psicose isquantified as in Example 111.

Example 119

To produce psicose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 80μM CoCl₂, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U P6PE,and 0.05 U P6PP is incubated at 50° C. for 24 hours. Production ofpsicose is quantified as in Example 111.

Example 120

To produce psicose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 80 μMCoCl2, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U P6PE,and 0.05 U P6PP is incubated at 50° C. for 24 hours. Production ofpsicose is quantified as in Example 111.

Example 121

To further increase yields of psicose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inexample 20. Production of psicose is quantified as in Example 111.

The invention includes all embodiments and variations substantially ashereinbefore described and with reference to the examples and figures.Although various embodiments of the invention are disclosed herein,adaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art.

What is claimed:
 1. A process for preparing mannose from a saccharide,the process comprising: converting fructose 6-phosphate (F6P) to mannose6-phosphate (M6P) catalyzed by mannose 6-phosphate isomerase (M6PI) orphosphoglucose/phosphomannose isomerase (PGPMI); and converting the M6Pto mannose catalyzed by mannose 6-phosphate phosphatase (M6PP).
 2. Theprocess of claim 1, further comprising a step of converting glucose6-phosphate (G6P) to the F6P, wherein the step is catalyzed by aphosphoglucoisomerase (PGI) or PGPMI.
 3. The process of claim 2, furthercomprising the step of converting glucose 1-phosphate (G1P) to the G6P,wherein the step is catalyzed by a phosphoglucomutase (PGM).
 4. Theprocess of claim 3, further comprising the step of converting asaccharide to the G1P, wherein the step is catalyzed by at least oneenzyme, wherein the saccharide is starch, a starch derivative, orsucrose.
 5. The process of claim 4, wherein the at least one enzyme inthe step of converting a saccharide to the G1P is selected from thegroup consisting of an alpha-glucan phosphorylase (αGP), a sucrosephosphorylase, and mixtures thereof.
 6. The process of claim 4, whereinthe saccharide is starch or a derivative thereof selected from the groupconsisting of amylose, amylopectin, soluble starch, amylodextrin,maltodextrin, maltose, and glucose, and mixtures thereof.
 7. The processof claim 6, further comprising the step of converting starch to a starchderivative wherein the starch derivative is prepared by enzymatichydrolysis of starch or by acid hydrolysis of starch.
 8. The process ofclaim 6, wherein 4-glucan transferase (4GT) is added to the process. 9.The process of claim 4, wherein the starch derivative is prepared byenzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase,alpha-amylase, or a combination thereof.
 10. The process of claim 1,wherein the process steps are conducted at a temperature ranging fromabout 37° C. to about 95° C., at a pH ranging from about 5.0 to about9.0, and/or for about 0.5 hours to about 48 hours.
 11. The process ofclaim 1, wherein the process steps are conducted in a single bioreactor.12. The process of claim 1, wherein the process steps are conductedATP-free, NAD(P)(H)-free, at a phosphate concentration from about 0.1 mMto about 150 mM, the phosphate is recycled, and/or the catalysis by thehexose-specific phosphatase involves an energetically favorable chemicalreaction.
 13. The process of claim 1, wherein the M6PI comprises anamino acid sequence having at least 25% sequence identity with any oneof SEQ ID Nos: 8-11, and wherein said M6PI catalyzes the conversion ofF6P to M6P.
 14. The process of claim 13, wherein the M6PI contains twodomains with a core of antiparallel β-strands resembling the cupin foldand a third domain consisting of only α-helixes, and a divalent metalcation.
 15. The process of claim 1, wherein the PGPMI comprises an aminoacid sequence having at least 25% sequence identity with any one of SEQID Nos: 15-17, and wherein said PGPMI catalyzes the conversion of F6P toM6P.
 16. The process of claim 15, wherein the PGPMI contains twoRossmanoid folds, a GGS motif, a SYSG-X-T-X-ET-hydrophobic motif, an ENsignature where the Glu is present for active-site base proton transfer,and an HN signature where the HIS is present for ring opening/closure ofthe substrate during catalysis.
 17. The process of claim 1, wherein theM6PP comprises an amino acid sequence having at least 30% sequenceidentity with any one of SEQ ID Nos: 12-14, and wherein said M6PPcatalyzes the conversion of M6P to mannose.
 18. The process of claim 17,wherein the M6PP contains a Rossmanoid fold domain for catalysis, a C1capping domain, D×D signature in the 1st β-strand of the Rossmanoidfold, a Thr or Ser at the end of the 2nd β-strand of the Rossmanoidfold, a Lys at the N-terminus of the α-helix C-terminal to the 3rdβ-strand of the Rossmanoid fold, and a GDxxxD signature at the end ofthe 4th β-strand of the Rossmanoid fold.